The Relationship between Cell Membrane Potassium Ion Transport and Glycolysis : The effect of ethacrynic acid

Cell membrane transport of K⁺ stimulates the rate of glycolysis in Ehrlich ascites tumor cells. A study of the characteristics of this relationship indicates that the stimulation occurs under anaerobic as well as under aerobic conditions. The data suggest that glycolysis is stimulated by a K⁺ transport mechanism that is coupled to Na⁺ transport because the effect is blunted or abolished when the principal intracellular ion is lithium or choline. This stimulus to glycolysis is blocked by ouabain and ethacrynic acid, agents that have been shown to inhibit monovalent cation transport in erythrocytes. In contrast to the action of ouabain, glycolysis is inhibited by ethacrynic acid in Ehrlich ascites tumor cells in the absence of cell membrane K⁺ transport. In studies with ghost-free hemolysates of human erythrocytes and with cytosol prepared from Ehrlich ascites tumor cells, ethacrynic acid significantly blocks lactate formation from fructose diphosphate demonstrating the direct inhibitory effect of this agent on one or more enzymes of the Embden-Meyerhof pathway. Since ethacrynic acid has no influence on lactate formation in intact erythrocytes utilizing an endogenous substrate, the presumptive site of inhibition is proximal to the 3-phosphoglycerate level.     

Lanthanum-induced alterations of cellular electrolytes in Ehrlich ascites tumor cells: a new view

We have shown previously that Ehrlich ascites tumor cells maintained at room temperature under an oxygen atmosphere lose Na⁺, K⁺ and Cl^? isosmotically when exposed to La⁺⁺⁺ (0.1 to 1.0 mM). Concomitant with these changes there is an increase in the recorded membrane potential (increasing intracellular negativity). The present studies further characterize the effect of La⁺⁺⁺ on electrolyte distribution. Ehrlich ascites tumor cells were maintained at 0.5° C to permit Na⁺ gain and K⁺ loss. The addition of 1 mM La⁺⁺⁺ to low temperature cells induces rapid loss of Na⁺, K⁺ and Cl^?. This net loss of cellular electrolytes occurs even in cells depleted of ATP content using 2-deoxyglucose (5 mM) and rotenone (10^?6 M ). Analysis of the appearance of tracer ²²Na in the environment of cells preloaded with the radioisotope shows that La⁺⁺⁺-induced changes in membrane permeability or in active ion transport mechanisms are not responsible for the dramatic loss of electrolytes from experimental cells. The electrolyte loss occurs only when the cells are resuspended mechanically during the washing procedure used to prepare the cells for electrolyte determination. We conclude that the results of La⁺⁺⁺ interaction with Ehrlich ascites tumor cells are twofold. As we have previously reported, La⁺⁺⁺ stabilizes and causes a hyperpolarization of the membrane potential. Secondly, La⁺⁺⁺ predisposes the cell membrane to become highly permeable when subjected to mechanical stress.     

Ca2+-dependent K+ transport in the Ehrlich ascites tumor cell

The possible presence and properties of the Ca²⁺-dependent K⁺ channel have been investigated in the Ehrlich ascites tumor cell. The treatment with ionophore A23187+Ca²⁺, propranolol or the electron donor system ascorbate-phenazine methosulphate, all of which activate that transport system in the human erythrocyte, produces in the Ehrlich cell a net loss of K⁺ (balanced by the uptake of Na⁺) and a stimulation of both the influx and the efflux of ⁸⁶Rb. These effects were antagonized by quinine, a known inhibitor of the Ca²⁺-dependent K⁺ channel in other cell systems, and by the addition of EGTA to the incubation medium. Ouabain did not have an inhibitory effect. These results suggests that the Ehrlich cell possesses a Ca²⁺-dependent K⁺ channel whose characteristics are similar to those described in other cell systems.     

The Na+ gradient hypothesis in cytoplasts derived from Ehrlich ascites tumor cells

The concentration gradients of Na⁺ and the non-metabolizable amino acid, α-aminoisobutyric acid, and the membrane potential were measured in cytoplasts derived from Ehrlich ascites tumor cells in order to test the Na⁺ gradient hypothesis for the active transport of neutral amino acids in animal cells. According to this hypothesis, the Na⁺ electrochemical gradient and the amino acid activity gradient should be equal at the steady state. It has been difficult to measure the Na⁺ electrochemical gradient in intact Ehrlich cells because Na⁺ may be sequestered in the nuclei of these cells. This problem is avoided with cytoplasts derived from Ehrlich cells because they do not contain internal compartments where Na⁺ could be sequestered. Since these cytoplasts also maintain steady state concentrations of Na⁺, K⁺, and α-aminoisobutyric acid similar to those found in whole Ehrlich cells, they are uniquely suited for testing the Na⁺ gradient hypothesis. Assuming the activity coefficients of external and cytoplasmic Na⁺ are equal, the energy in the Na⁺ electrochemical gradient of cytoplasts was 90% of that in the α-aminoisobutyric acid concentration gradient at the steady state. If the Na⁺ gradient hypothesis is correct, the 10% difference between these two gradients cannot be explained in terms of the sequestration of Na⁺ in the nucleus because cytoplasts do not contain internal compartments.     

The preparation and properties of cytoplasts from Ehrlich ascites tumor cells

A method is described in which cytochalasin B is used to fractionate Ehrlich ascites tumor cells into cytoplasts and (nucleated) karyoplasts. The plasma membrane and cytoplasm are selectively removed from these cells by this method such that the cytoplasts rarely contain membranous organelles (e.g., mitochondria) which are retained in the karyoplast during fractionation. ATP concentrations similar to those found in whole cells and glycolytic activity were measured in cytoplasts in the presence but not the absence of glycose. Cytoplasts also actively transport Na⁺, K⁺, and α-aminoisobutyric acid to steadystate distribution ratios similar to those found in whole cells. It was concluded that these cytoplasts are a simplified model system for the study of active transport in Ehrlich cells.     

Phosphate transport and its relationship to cation movements in Ehrlich Lettré ascites tumor cells.

In view of the importance of P_i in the control of cell metabolism, it was of interest to study the mechanism and regulation of P_i uptake by ascites tumor cells. For this purpose, the incorporation of ³²P_i into Ehrlich Lettré cells was compared when competitive anions and inhibitors which alter cation movements were present. Anions such as sulfanilate (35 mm) and succinate (30 mm) decrease ³²P_i uptake by ca. 35%, suggesting that transport is mediated by a protein similar to the 100,000 M_r anion carrier isolated from erythrocyte membranes. Furosemide, a diuretic which bears a structural analogy to sulfanilate inhibitors of anion transport, also decreases ³²P_i incorporation at concentrations as low as 2 × 10^?5m. This inhibitor blocks cation exchange in ascites tumor cells, and from the present data, it is suggested that a possible function of the furosemidesensitive cation exchange protein is to facilitate anion transport. Ouabain, known to inhibit (Na⁺ + K⁺)-ATPase and its dephosphorylation, stimulates the rate of incorporation of ³²P_i into cells and also raises the net inorganic phosphate level. The stimulation of ³²P_i incorporation is decreased by sulfanilate or succinate. In contrast to the effects of ouabain, addition of 10 mm K⁺, which is known to stimulate (Na⁺ + K⁺)-ATPase and its dephosphorylation, decreases ³²P_i incorporation. These observations suggest that anion transport and energy-dependent Na⁺ and K⁺ movements may be closely coupled to the intact cell.     

Studies on the relationship between glycolysis and (Na++K+)-ATPase in cultured cells

In several tissues a coupling between glycolysis and (Na⁺+K⁺)-ATPase has been observed. We report here studies on the coupling of glycolysis and (Na⁺+K⁺)-ATPase in Rous-transformed hamster cells and Ehrlich ascites tumor cells. The rate of (Na⁺+K⁺)-ATPase was estimated by the initial rate of ouabain-sensitive K⁺ influx after K⁺ reintroduction to K⁺-depleted cells. Experiments were performed with cells producing ATP via oxidative phosphorylation alone (i.e., lactate sole substrate), glycolysis alone (i.e., glucose as substrate in the absence of oxygen or with antimycin A), or glycolysis and oxidative phosphorylation (i.e., glucose as substrate in the presence of oxygen). The cells produced ATP at approximately the same rate under all of these conditions, but the initial rate of K⁺-influx was approx. 2-fold higher when AtP was produced from glycolysis. Changes in cell Na⁺ due to other transport processes related to glycolysis, such as Na⁺-H⁺ exchange, Na⁺-glucose cotransport, and K⁺-H⁺ exchange were ruled out as mediators of this effect on (Na⁺+K⁺)-ATPase. These data suggest that glycolysis is more effective than oxidative phosphorylation in providing ATP to (Na⁺+K⁺)-ATPase to these cultured cells.     

Contribution of intracellular ATP to cisplatin resistance of tumor cells

Decreased cellular accumulation of cisplatin is a frequently observed mechanism of resistance to the drug. Beside passive diffusion, several cellular proteins using ATP hydrolysis as an energy source are assumed to be involved in cisplatin transport in and out of the cell. This investigation aimed at clarifying the contribution of intracellular ATP as an indicator of energy-dependent transport to cisplatin resistance using the A2780 human ovarian adenocarcinoma cell line and its cisplatin-resistant variant A2780cis. Depletion of intracellular ATP with oligomycin significantly decreased cellular platinum accumulation (measured by flameless atomic absorption spectrometry) in sensitive but not in resistant cells, and did not affect cisplatin efflux in both cell lines. Inhibition of Na⁺,K⁺-ATPase with ouabain reduced platinum accumulation in A2780 cells but to a lesser extent compared with oligomycin. Western blot analysis revealed lower expression of Na⁺,K⁺-ATPase α₁ subunit in resistant cells compared with sensitive counterparts. The basal intracellular ATP level (determined using a bioluminescence-based assay) was significantly higher in A2780cis cells than in A2780 cells. Our results highlight the importance of ATP-dependent transport, among other processes mediated by Na⁺,K⁺-ATPase, for cisplatin influx in sensitive cells. Cellular platinum accumulation in resistant cells is reduced and less dependent on energy sources, which may partly result from Na⁺,K⁺-ATPase downregulation. Our data suggest the involvement of other ATP-dependent processes beside those regulated by Na⁺,K⁺-ATPase. Higher basal ATP level in cisplatin-resistant cells, which appears to be a consequence of enhanced mitochondrial ATP production, may represent a survival mechanism established during development of resistance.     

The effect of the fluorescent probe, 3,3′-dipropylthiodicarbocyanine iodide,on the membrane potential of Ehrlich ascites tumor cells

The effects of the potential-sensitive fluorescent dye, 3,3′dipropylthiodicarbocyanine iodide, on factors establishing the membrane potential of Ehrlich ascites tumor cells have been tested. The dye itself induces membrane hyperpolarization as monitored by electrophysiological methods. In addition, the dye inhibits active (Na⁺+K⁺-transport and increases cell membrane permeability to K⁺ by about 65% in these cells.     

Neutral amino acid transport in surface membrane vesicles isolated from mouse fibroblasts: Intrinsic and extrinsic models of regulation

Membrane transport carrier function, its regulation and coupling to metabolism, can be selectively investigated dissociated from metabolism and in the presence of a defined electrochemical ion gradient driving force, using the single internal compartment system provided by vesiculated surface membranes. Vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated transport of several neutral amino acids into an osmotically-sensitive intravesicular space without detectable metabolic conversion of substrate. When a Na⁺ gradient, external Na⁺ > internal Na⁺, was artifically imposed across vesicle membranes, accumulation of several neutral amino acids achieved apparent intravesicular concentrations 6- to 9-fold above their external concentrations. Na⁺-stimulated alanine transport activity accompanied plasma membrane material during subcellular fractionation procedures. Competitive interactions among several neutral amino acids for Na⁺-stimulated transport into vesicles and inactivation studies indicated that at least 3 separate transport systems with specificity properties previously defined for neutral amino acid transport in Ehrlich ascites cells were functional in vesicles from mouse fibroblasts: the A system, the L system and a glycine transport system. The pH profiles and apparent K_m values for alanine and 2-aminoisobutyric acid transport into vesicles were those expected of components of the corresponding cellular uptake system. Several observations indicated that both a Na⁺ chemical concentration gradient and an electrical membrane potential contribute to the total driving force for active amino acid transport via the A system and the glycine system. Both the initial rate and quasi-steady-state of accumulation were stimulated as a function of increasing concentrations of Na⁺ applied as a gradient (external > internal) across the membrane. This stimulation was independent of endogenous Na⁺, K⁺-ATPase activity in vesicles and was diminished by monensin or by preincubation of vesicles with Na⁺. The apparent K_m for transport of alanine and 2-aminoisobutyric acid was decreased as a function of Na⁺ concentration. Similarly, in the presence of a standard initial Na⁺ gradient, quasi-steady-state alanine accumulation in vesicles increased as a function of increasing magnitudes of interior-negative membrane potential imposed across the membrane by means of K⁺ diffusion potentials (internal > external) in the presence of valinomycin; the magnitude of this electrical component was estimated by the apparent distributions of the freely permeant lipophilic cation triphenylme thylphosphonium ion. Alanine transport stimulation by charge asymmetry required Na⁺ and was blocked by the further addition of either nigericin or external K⁺. As a corollary, Na⁺-stimulated alanine transport was associated with an apparent depolarization, detectable as an increased labeled thiocyanate accumulation. Permeant anions stimulated Na⁺-coupled active transport of these amino acids but did not affect Na⁺-independent transport. Translocation of K⁺, H⁺, or anions did not appear to be directly involved in this transport mechanism. These characteristics support an electrogenic mechanism in which amino acid translocation is coupled t o an electrochemical Na⁺ gradient by formation of a positively charged complex, stoichiometry unspecified, of Na⁺, amino acid, and membrane component. Functional changes expressed in isolated membranes were observed t o accompany a change in cellular proliferative state or viral transformation. Vesicles from Simian virus 40-transformed cells exhibited an increased Vmax of Na⁺-stimulated 2-aminoisobutyric acid transport, as well as an increased capacity for steady-state accumulation of amino acids in response t o a standard Na⁺ gradient, relative t o vesicles from nontransformed cells. Density-inhibition of nontransformed cells was associated with a marked decrease in these parameters assayed in vesicles. Several possibilities for regulatory interactions involving gradient-coupled transport systems are discussed.     

The effect of reversal of Na and K electrochemical potential gradients on the active transport of amino acids in Ehrlich ascites tumor cells

1.

1. The net uptake of α-aminoisobutyric acid (AIB) in Ehrlich ascites tumor cells has been studied under a variety of transmembrane concentration gradients of Na⁺, K⁺ and AIB itself.     

Cell detachment in ouabain-sensitive and resistant ehrlich ascites cells

Ehrlich ascites tumor cells selected for resistance (OR) to the toxic effects of ouabain, a specific inhibitor of (Na⁺, K⁺)-activated ATPase, differ from wild-type ouabain-sensitive (OS) ascites cells in their detachment behavior from protein-coated glass surfaces in the absence of ouabain. In the presence of ouabain OS cells are more easily detached from cultured vessel surfaces, but ouabain is either without effect (10⁻⁴ M) or inhibits detachment of OR cells (10⁻³ M) under similar conditions. The results are discussed in terms of the possible relationship between membrane ATPases and cell behavior.     

Regulation of the electrogenic (Na+ + K+)-pump of Ehrlich cells by intracellular cation levels

Ehrlich cells actively accumulate neutral amino acids even if both the Na⁺ and K⁺ gradients are inverted. The seeming contradiction of this observation to the gradient hypothesis is, however, explained by the presence of a powerful electrogenic Na⁺ pump, which stongly raises the electrochemical potential gradient of Na⁺ under these conditions. Since the evidence of this pump has so far been found only during abnormal concentrations of alkali ions (low K⁺, high Na⁺) in these cells, the question arises whether the pump is equally powerful with completely normal cells, when the pump is not ‘needed’ for amino acid transport. Using the initial rate of uptake of the test amino acid (2-aminoisobutyrate) as a sensitive monitor of the electrical potential at constant cation distribution between cell and medium, a procedure has been devised to split the overall electrical potential into the diffusional and the pump component. With this procedure it could be shown that the electrogenic pump per se is most powerful in K⁺-depleted and Na⁺-rich cells but declines to a lower ‘resting’ value according as the electrolyte content of the cell approaches normality. A strong positive correlation between cellular Na⁺ content and the electrogenic pumping activity suggests that the intracellular activity of this ion regulates the rate of the electrogenic pump. The low activity of the pump under normal conditions may explain why the existance of this pump has rarely come to attention previously.     

Effects of valinomycin,ouabain, and potassium on glycolysis and intracellular pH of Ehrlich ascites tumor cells

Summary Both valinomycin and ouabain block reaccumulation of K⁺ by Ehrlich ascites tumor cells depleted of K⁺ and cause loss of K⁺ from high-K⁺ cells. Glucose largely reverses the effect of valinomycin and to a lesser extent that of ouabain.In cells depleted of K⁺, glucose utilization and lactate production are impaired. Neither extracellular pH (pH_e) nor intracellular pH (pH_i) falls to the extent seen in non-depleted glycolyzing cells. Addition of K⁺ to depleted cells reverses these effects. Valinomycin increases glycolysis in K⁺-depleted cells but to a greater extent in nondepleted or K⁺-repleted cells. The increase in lactate production caused by valinomycin is accompanied by a correspondingly greater fall in pH_e and pH_i. Valinomycin, unlike other uncoupling agents, does not abolish the pH gradient across the plasma membrane. Increased utilization of glucose resulting from addition of K⁺ to K⁺-depleted cells or addition of valinomycin either to depleted or non-depleted cells can be entirely accounted for by increased lactate production. Ouabain blocks the stimulatory effect of added K⁺ on K⁺-depleted cells and has an inhibitory effect on glycolysis in non-depleted cells. It does not obliterate the difference in glycolytic activity between K⁺-depleted and nondepleted cells. Ouabain does not completely block the effect of valinomycin in augmenting glycolysis in depleted or non-depleted cells. Increased accumulation of glycolytic intermediates, particularly dihydroxyacetone phosphate, is found in glycolyzing K⁺-depleted cells. The most marked accumulation was found in ouabain-treated K⁺-deficient cells.This investigation was supported by U.S. Public Health Service grant no. GM 13606 from the National Institute of General Medical Sciences and by grant no. P-603 from the American Cancer Society.PHS Research Career Program Award 4 K06 GM 19429 from the National Institute of General Medical Sciences.     

Electrically silent cotransport of Na+, K+ and Cl− in ehrlich cells

A cotransport system for Na⁺, K⁺ and Cl^? in Ehrlich cells is described. It is insensitive towards ouabain but specifically inhibited by furosemide and other ‘high ceiling’ diuretics at concentrations which do not affect other pathways of the ions concerned. As the furosemide-sensitive fluxes of these ions are not affected by changes in membrane potential, and as their complete inhibition by furosemide does not appreciably alter the membrane potential, they appear to be electrically silent. Application of the pulse-response methods in terms of irreversible thermodynamics reveals tight coupling between the furosemide-sensitive flows of Na⁺, K⁺ and Cl^? ($\text{q}$ close to unity for all three combinations) at a stoichiometry of 1 : 1 : 2. The site for each of the ions appears to be rather specific: K⁺ can be replaced by Rb⁺ but not by other cations tested whereas Cl^? can be poorly replaced by Br^? but not by NO₃^?, in contradistinction to the Cl^?-OH^? exchange system. The cotransport system appears to function in cell volume regulation as it tends to make the cell swell, thus counteracting the shrinking effect of the ouabain-sensitive (Na⁺, K⁺) pump.The experiments presented could not clarify whether the cotransport process is a primary or secondary active one; while incongruence between transport and conjugated driving force seems to indicate primary active transport, it is very unlikely that hydrolysis of ATP supplies energy for the transport process, since there is no stimulation of ATP turnover observable under operation of the cotransport system.     

Salt-inducible kinase 1 is present in lung alveolar epithelial cells and regulates active sodium transport

Salt-inducible kinase 1 (SIK1) in epithelial cells mediates the increases in active sodium transport (Na⁺, K⁺-ATPase-mediated) in response to elevations in the intracellular concentration of sodium. In lung alveolar epithelial cells increases in active sodium transport in response to β-adrenergic stimulation increases pulmonary edema clearance. Therefore, we sought to determine whether SIK1 is present in lung epithelial cells and to examine whether isoproterenol-dependent stimulation of Na⁺, K⁺-ATPase is mediated via SIK1 activity. All three SIK isoforms were present in airway epithelial cells, and in alveolar epithelial cells type 1 and type 2 from rat and mouse lungs, as well as from human and mouse cell lines representative of lung alveolar epithelium. In mouse lung epithelial cells, SIK1 associated with the Na⁺, K⁺-ATPase α-subunit, and isoproterenol increased SIK1 activity. Isoproterenol increased Na⁺, K⁺-ATPase activity and the incorporation of Na⁺, K⁺-ATPase molecules at the plasma membrane. Furthermore, those effects were abolished in cells depleted of SIK1 using shRNA, or in cells overexpressing a SIK1 kinase-deficient mutant. These results provide evidence that SIK1 is present in lung epithelial cells and that its function is relevant for the action of isoproterenol during regulation of active sodium transport. As such, SIK1 may constitute an important target for drug discovery aimed at improving the clearance of pulmonary edema.     

Effects of nucleotides and Na + on ouabain activation of the phosphatase associated with Na + , K + -ATPase

Ouabain activation of the phosphatase associated with Na⁺,K⁺-ATPase is a time-dependent process which is stimulated by ATP and other nucleotides. Further stimulation by Na⁺ is observed under certain conditions. The stimulatory effect of ATP was found to be due to an increase in the affinity of the enzyme for ouabain. The time required for maximal ouabain activation to be achieved was decreased by ATP and further decreased by ATP + Na⁺.These conditions for maximal activation by ouabain are similar to those required for maximal ouabain binding and suggest that the same ouabain site is responsible for activation of Mg²⁺-dependent phosphatase and for inhibition of Na⁺,K⁺-ATPase and K⁺-phosphatase.     

Intracellular compartmentation of Na+, K+ and Cl− in the Ehrlich ascites tumor cell: Correlation with the membrane potential

Summary The intracellular distribution of Na⁺, K⁺, Cl^– and water has been studied in the Ehrlich ascites tumor cell. Comparison of the ion and water contents of whole cells with those of cells exposed to La³⁺ and mechanical stress indicated that La³⁺ treatment results in selective damage to the cell membrane and permits evaluation of cytoplasmic and nuclear ion concentrations. The results show that Na⁺ is sequestered within the nucleus, while K⁺ and Cl^– are more highly concentrated in the cell cytoplasm. Reduction of the [Na⁺] of the incubation medium by replacement with K⁺ results in reduced cytoplasmic [Na⁺], increased [Cl^–] and no change in [K⁺]. Nuclear concentrations of these ions are virtually insensitive to the cation composition of the medium. Concomitant measurements of the membrane potential were made. The potential in control cells was –13.7 mV. Reduction of [Na⁺] in the medium caused significant depolarization. The measured potential is describable by the Cl^– equilibrium potential and can be accounted for in terms of cation distributions and permeabilities. The energetic implications of the intracellular compartmentation of ions are discussed.     

Effects of extremely-law-frequency electromagnetic fields on ion transport in several mammalian cells

We have investigated the effects of sinusoidal electromagnetic fields (EMF) on ion transport (Ca²⁺, Na⁺, K⁺, and H⁺) in several cell types (red blood cells, thymocytes, Ehrlich ascites tumor cells, and HL60 and U937 human leukemia cells). The effects on the uptake of radioactive tracers as well as on the cytosolic Ca²⁺ concentration ([Ca²⁺]_i), the intracellular pH (pH_i), and the transmembrane potentsial (TMP) were studied. Exposure to EMF at 50 Hz and 100–2000 μT (rms) had no significant effects on any of these parameters. Exposure to EMF of 20–1200 μT (rms) at the estimated cyclotron magnetic resonance frequencies for the respective ions had no significant effects except for a 12–32% increase of the uptake of ⁴²K within a window at 14.5–15.5 Hz and 100–200 μT (rms), which was found in U937 and Ehrlich cells but not in the other cell types. © 1994 Wiley-Liss, Inc.     

Inhibition of K+ Transport through Na+, K+-ATPase by Capsazepine: Role of Membrane Span 10 of the α-Subunit in the Modulation of Ion Gating

Capsazepine (CPZ) inhibits Na⁺,K⁺-ATPase-mediated K⁺-dependent ATP hydrolysis with no effect on Na⁺-ATPase activity. In this study we have investigated the functional effects of CPZ on Na⁺,K⁺-ATPase in intact cells. We have also used well established biochemical and biophysical techniques to understand how CPZ modifies the catalytic subunit of Na⁺,K⁺-ATPase. In isolated rat cardiomyocytes, CPZ abolished Na⁺,K⁺-ATPase current in the presence of extracellular K⁺. In contrast, CPZ stimulated pump current in the absence of extracellular K⁺. Similar conclusions were attained using HEK293 cells loaded with the Na⁺ sensitive dye Asante NaTRIUM green. Proteolytic cleavage of pig kidney Na⁺,K⁺-ATPase indicated that CPZ stabilizes ion interaction with the K⁺ sites. The distal part of membrane span 10 (M10) of the α-subunit was exposed to trypsin cleavage in the presence of guanidinum ions, which function as Na⁺ congener at the Na⁺ specific site. This effect of guanidinium was amplified by treatment with CPZ. Fluorescence of the membrane potential sensitive dye, oxonol VI, was measured following addition of substrates to reconstituted inside-out Na⁺,K⁺-ATPase. CPZ increased oxonol VI fluorescence in the absence of K⁺, reflecting increased Na⁺ efflux through the pump. Surprisingly, CPZ induced an ATP-independent increase in fluorescence in the presence of high extravesicular K⁺, likely indicating opening of an intracellular pathway selective for K⁺. As revealed by the recent crystal structure of the E₁.AlF₄ ^-.ADP.3Na⁺ form of the pig kidney Na⁺,K⁺-ATPase, movements of M5 of the α-subunit, which regulate ion selectivity, are controlled by the C-terminal tail that extends from M10. We propose that movements of M10 and its cytoplasmic extension is affected by CPZ, thereby regulating ion selectivity and transport through the K⁺ sites in Na⁺,K⁺-ATPase.