The mechanism of insulin stimulation of (Na+,K+)-ATPase transport activity in muscle

Since the mechanism underlying the insulin stimulation of (Na+,K+)-ATPase transport activity observed in multiple tissues has remained undetermined, we have examined (Na+,K+)-ATPase transport activity (ouabain-sensitive 86Rb+ uptake) and Na+/H+ exchange transport (amiloride-sensitive 22Na+ influx) in differentiated BC3H-1 cultured myocytes as a model of insulin action in muscle. The active uptake of 86Rb+ was sensitive to physiological insulin concentrations (1 nM), yielding a maximum increase of 60% without any change in 86Rb+ permeability. In order to determine the mechanism of insulin stimulation of (Na+,K+)-ATPase activity, we demonstrated that insulin also stimulates passive 22Na+ influx by Na+/H+ exchange transport (maximal 200% increase) and an 80% increase in intracellular Na+ concentration with an identical time course and dose-response curve as insulin-stimulated (Na+,K+)-ATPase transport activity. Incubation of the cells with high [Na+] (195 mM) significantly potentiated insulin stimulation of ouabain-inhibitable 86Rb+ uptake. The ionophore monensin, which also promotes passive Na+ entry into BC3H-1 cells, mimics the insulin stimulation of ouabain-inhibitable 86Rb+ uptake. In contrast, incubation with amiloride or low [Na+] (10 mM), both of which inhibit Na+/H+ exchange transport, abolished the insulin stimulation of (Na+,K+)-ATPase transport activity. Furthermore, each of these insulin-stimulated transport activities displayed a similar sensitivity to amiloride. These results indicate that insulin stimulates a large increase in Na+/H+ exchange transport and that the resulting Na+ influx increases the intracellular Na+ concentration, thus activating the internal Na+ transport sites of the (Na+,K+)-ATPase. This Na+ influx is, therefore, the mediator of the insulin-induced stimulation of membrane (Na+,K+)-ATPase transport activity classically observed in muscle.     

A bar model for the pump and channel function of the reconstituted Na+,K+-ATPase

Purified Na+,K+-ATPase is treated with trypsin. The altered enzyme is then reconstituted into liposomes and the change in active and passive Na+,K+-fluxes is recorded. Trypsin treatment transforms the slow passive Na+,K+-fluxes into leaks. The leak formation is correlated with the degree of proteolysis and the associated decrease in Na+,K+-ATPase activity. The active Na+,K+-transport capacity decreases in parallel with the passive transport. It is thus proposed that the Na+,K+-ATPase molecule primarily contains unspecific transmembrane tunnels that are rendered ion-selective by transverse bars of specific length (bar model).     

A reconstituted Na+ + K+ pump in liposomes containing purified (Na+ + K+)-ATPase from kidney medulla.

Liposomes containing either purified or microsomal (Na+,K+)-ATPase preparations from lamb kidney medulla catalyzed ATP-dependent transport of Na+ and K+ with a ratio of approximately 3Na+ to 2K+, which was inhibited by ouabain. Similar results were obtained with liposomes containing a partially purified (Na+,K+)-ATPase from cardiac muscle. This contrasts with an earlier report by Goldin and Tong (J. Biol. Chem. 249, 5907-5915, 1974), in which liposomes containing purified dog kidney (Na+,K+)-ATPase did not transport K+ but catalyzed ATP-dependent symport of Na+ and Cl-. When purified by our procedure, dog kidney (Na+,K+)-ATPase showed some ability to transport K+ but the ratio of Na+ : K+ was 5 : 1.     

Modulation of gastric H+,K+-transporting ATPase function by sodium

Gastric H+,K+-ATPase activity is not affected by Na+ at pH 7.0 but is significantly stimulated by Na+ at pH 8.5. For the stimulation at the latter pH, the presence of both Na+ and K+ were essential. Contrary the H+,K+-ATPase, the associated K+-pNPPase was inhibited by Na+ at both pH values. Sodium competes with K+ for the K+-pNPPase reaction. Also, unlike the H+, K+-ATPase activity the ATPase-mediated transport of H+ within the gastric microsomal vesicles was inhibited by Na+. For the latter event only the extravesicular and not the intravesicular Na+ was effective. The data suggest that the K+-pNPPase activity does not represent the phosphatase step of the H+,K+-ATPase reaction. In addition, the observed inhibition of vesicular H+ uptake by Na+ appears to be due to the displacement by Na+ of a cytosolic (extravesicular) H+ site responsible for the vectorial translocation of H+.     

Sodium-potassium adenosine triphosphatase activity of human lymphocyte membrane vesicles: kinetic parameters, substrate specificity, and effects of phytohemagglutinin.

We have prepared human blood lymphocyte membrane vesicles of high purity in sufficient quantity for detailed enzyme analysis. This was made possible by the use of plateletpheresis residues, which contain human lymphocytes in amounts equivalent to thousands of milliliters of blood. The substrate specificity and the kinetics of the cofactor and substrate requirements of the human lymphocyte membrane Na+, K+-ATPase activity were characterized. The Na+, K+-ATPase did not hydrolyze ADP, AMP, ITP, UTP, GTP or TTP. The mean ATPase stimulated by optimal concentrations of Na+ and K+ (Na+, K+-ATPase) was 1.5 nmol of P(i) hydrolyzed, microgram protein-1, 30 min-1 (range 0.9-2.1). This activity was completely inhibited by the cardiac glycoside, ouabain. The K(m) for K+ was approximately 1.0 mM and the K(m) for Na+ was approximately 15 mM. Active Na+ and K+ transport and ouabain-sensitive ATP production increase when lymphocytes are stimulated by PHA. Na+, K+-ATPase activity must increase also to transduce energy for the transport of Na+ and K+. Some studies have reported that PHA stimulates the lymphocyte membrane ATPase directly. We did not observe stimulation of the membrane Na+, K+-ATPase when either lymphocytes or lymphocyte membranes were treated with mitogenic concentrations of PHA. Moreover, PHA did not enhance the reaction velocity of the Na+, K+-ATPase when studied at the K(m) for ATP, Na+, K+ OR Mg++, indicating that it does not alter the affinity of the enzyme for its substrate or cofactors. Thus, our data indicate that the increase in ATPase activity does not occur as a direct result of PHA action on the cell membrane.     

Na+,K+-ATPase: structure, mechanism, and regulation

Structural organization of alpha- and beta-subunits of Na+,K+-ATPase in the membrane, the enzyme oligomeric structure, and mechanisms of ATP hydrolysis and cation transport are considered. The data on the structure of cation-binding sites and ion-conductive pathways of the pump are reviewed. The properties of isoforms of both subunits are described. Special attention was paid to the ATP modifying effect on Na+,K+-ATPase. To explain the rather complex dependence of the Na+,K+-ATPase activity on ATP concentration, a hypothesis is proposed, which is based on the assumption that the membrane contains the enzyme protomer exhibiting high affinity to ATP and an oligomer having low affinity to the nucleotide and characterized by positive cooperative interactions between subunits. Data on the Na+,K+-ATPase phosphorylation by protein kinases A and C are reviewed.     

Absence of effect of aldosterone on sodium efflux catalyzed by the human erythrocyte Na+, K+-ATPase in vitro

The effect of physiological and pharmacological concentrations of aldosterone on Na+ efflux catalyzed by the human erythrocyte Na+,K+-ATPase in vitro were studied. Aldosterone had no significant effect on ouabain-sensitive Na+ efflux from fresh erythrocytes. In addition, aldosterone did not alter Na+ transport activity of stimulated Na+,K+-ATPase of Na+ loaded erythrocytes. Finally, Na+ efflux from Na+ loaded erythrocytes was not changed by preincubation of the cells with aldosterone. It is concluded that aldosterone in vitro does not modify pump activity of the human erythrocyte Na+, K+-ATPase.     

Regulation by aldosterone of Na+,K+-ATPase mRNAs, protein synthesis, and sodium transport in cultured kidney cells

Transepithelial Na+ reabsorption across tight epithelia is regulated by aldosterone. Mineralocorticoids modulate the expression of a number of proteins. Na+,K+-ATPase has been identified as an aldosterone-induced protein (Geering, K., M. Girardet, C. Bron, J. P. Kraehenbuhl, and B. C. Rossier, 1982, J. Biol. Chem., 257:10338-10343). Using A6 cells (kidney of Xenopus laevis) grown on filters we demonstrated by Northern blot analysis that the induction of Na+,K+-ATPase was mainly mediated by a two- to fourfold accumulation of both alpha- and beta-subunit mRNAs. The specific competitor spironolactone decreased basal Na+ transport, Na+,K+-ATPase mRNA, and the relative rate of protein biosynthesis, and it blocked the response to aldosterone. Cycloheximide inhibited the aldosterone-dependent sodium transport but did not significantly affect the cytoplasmic accumulation of Na+,K+-ATPase mRNA induced by aldosterone.     

Early and delayed changes in potassium transport during the initiation of cell proliferation in CHO culture

Stimulation by serum of cell proliferation in G1-arrested culture of Chinese hamster ovary cells CHO-K1 was accompanied by an early (during the first minutes) and delayed (2-10 h) activation of Na+,K+-ATPase and an increase in cell K+ content from 0.5-0.6 to 0.7-0.8 mmol per gram protein. Isoproterenol acted synergistically with serum in eliciting both early and delayed changes in K+ transport and in stimulating G1----S transition. Isoproterenol alone (without serum) induced a transient increase in K+ influx via Na+,K+-ATPase without changing the cell K+ content or having any mitogenic effect. Theophylline enhanced the serum-induced early activation of Na+,K+-ATPase but inhibited both the delayed increase in cell K+ and the G1----S transition. Early serum-induced increase in K+ transport was not affected by cycloheximide, whereas net accumulation of cell K+ was abolished by the drug. It is concluded that the early and the delayed activation of Na+,K+-ATPase induced by mitogens can be dissociated; the early ionic response is related to the primary transduction of membrane signal, whereas the delayed modulation of ion transport via Na+,K+-ATPase has another function and is associated with cell growth.     

(Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in kidney of sea bass (Dicentrarchus labrax L.)

1. Sea bass kidney microsomal preparations contain two Mg2+ dependent ATPase activities: the ouabain-sensitive (Na+ + K+)-ATPase and an ouabain-insensitive Na+-ATPase, requiring different assay conditions. The (Na+ + K+)-ATPase under the optimal conditions of pH 7.0, 100 mM Na+, 25 mM K+, 10 mM Mg2+, 5 mM ATP exhibits an average specific activity (S.A.) of 59 mumol Pi/mg protein per hr whereas the Na+-ATPase under the conditions of pH 6.0, 40 mM Na+, 1.5 mM MgATP, 1 mM ouabain has a maximal S.A. of 13.9 mumol Pi/mg protein per hr. 2. The (Na+ + K+)-ATPase is specifically inhibited by ouabain and vanadate; the Na+-ATPase specifically by ethacrynic acid and preferentially by frusemide; both activities are similarly inhibited by Ca2+. 3. The (Na+ + K+)-ATPase is specific for ATP and Na+, whereas the Na+-ATPase hydrolyzes other substrates in the efficiency order ATP greater than GTP greater than CTP greater than UTP and can be activated also by K+, NH4+ or Li+. 4. Minor differences between the two activities lie in the affinity for Na+, Mg2+, ATP and in the thermosensitivity. 5. The comparison between the two activities and with what has been reported in the literature only partly agree with our findings. It tentatively suggests that on the one hand two separate enzymes exist which are related to Na+ transport and, on the other, a distinct modulation in vivo in different tissues.     

Inactivation of Na+, K+ -ATPase from cattle brain by sodium fluoride

The influence of the physiological ligands and modifiers on the plasma membrane Na+, K+ -ATPase from calf brain inactivation by sodium fluoride (NaF) is studied. ATP-hydrolyzing activity of the enzyme was found to be more stable as to NaF inhibition than its K+ -pNPPase activity. The activatory ions of Na+, K+ -ATPase have different effects on the process of the enzyme inhibition by NaF. K+ intensifies inhibition, but Na+ does not affect it. An increase of [Mg2+free] in the incubation medium (from 0.5 to 3.0 mM) rises the sensitivity of Na+, K+ -ATPase to NaF inhibition. But an increase of [ATP] from 0.3 to 1.5 mM has no effect on this process. Ca and Mg ions modify Na+, K+ -ATPase inhibition by fluoride differently. Ca2+free levels this process, and Mg2+free on the contrary increases it. In the presence of Ca ions and in the neutral-alkaline medium (pH 7.0-8.5) the recovery of activity of the transport ATPase inhibited by-NaF takes place. Sodium citrate also protects both ATP-hydrolizing and K-pNPPase activity of the Na+, K+ -ATPase from NaF inhibition. Under the modifing membranous effects (the treatment of plasma membranes by Ds-Na and digitonin) the partial loss of Na+, K+ -ATPase sensitivity to NaF inhibition is observed. It is concluded that Na+, K+ -ATPase inactivation by NaF depends on the influence of the physiological ligands and modifiers as well as on the integrity of membrane structure.     

Dog kidney (Na+,K+)-ATPase is more sensitive to inhibition by vanadate than human red cell (Na+,K+)-ATPase

(Na+,K+)-ATPase (EC 3.6.1.3) from kidney is more sensitive to inhibition by vanadate than red cell (Na+,K+)-ATPase. The difference appears to be in the apparent affinities of the two enzymes for K+ and Na+ at sites where K+ promotes and Na+ opposes vanadate binding. As a result of Na+-K+ competition at these sites, reversal of vanadate inhibition was accomplished at lower Na+ concentrations in red cell than in kidney (NA+,K+)-ATPase. It is possible that vanadate could selectively regulate Na+ transport in the kidney.     

The energy transduction mechanism is different among P-type ion-transporting ATPases. Acetyl phosphate causes uncoupling between hydrolysis and ion transport in H+,K(+)-ATPase.

H+,K(+)-ATPase, Na+,K(+)-ATPase, and Ca(2+)-ATPase belong to the P-type ATPase group. Their molecular mechanisms of energy transduction have been thought to be similar until now. Ca(2+)-ATPase and Na+,K(+)-ATPase are phosphorylated from both ATP and acetyl phosphate (ACP) and dephosphorylated, resulting in active ion transport. However, we found that H+,K(+)-ATPase did not transport proton nor K+ when ACP was used as a substrate, resulting in uncoupling between energy and ion transport. ACP bound competitively to the ATP-binding site of H+,K(+)-ATPase. The hydrolysis of ACP by H+,K(+)-ATPase was stimulated by cytosolic K+, the half-maximal stimulating K+ concentration (K0.5) being 2.5 mM, whereas the hydrolysis of ATP was stimulated by luminal K+, the K0.5 being 0.2 mM. Furthermore, during the phosphorylation from ACP in the absence of K+, the fluorescence intensity of H+,K(+)-ATPase labeled with fluorescein isothiocyanate increased, but those of Na+,K(+)-ATPase and Ca(2+)-ATPase decreased. These results indicate that phosphorylated intermediates of H+,K(+)-ATPase formed from ACP are not rich in energy and that there is a striking difference(s) in the mechanism of energy transduction between H+,K(+)-ATPase and other cation-transporting ATPases.     

Na+-dependent amino acid transport is a major factor determining the rate of (Na+,K+)-ATPase mediated cation transport in intact HeLa cells

Little is known concerning the effects of Na+-coupled solute transport on (Na+,K+)-ATPase mediated cation pumping in the intact cell. We investigated the effect of amino acid transport and growth factor addition on the short term regulation of (Na+,K+)-ATPase cation transport in HeLa cells. The level of pump activity in the presence of amino acids or growth factors was compared to the level measured in phosphate buffered saline. These rates were further related to the maximal pump capacity, operationally defined as ouabain inhibitable 86Rb+ influx in the presence of 15 microM monensin. Of the growth factors tested, only insulin was found to moderately (22%) increase (Na+,K+)-ATPase cation transport. The major determinant of pump activity was found to be the transport of amino acids. Minimal essential medium (MEM) amino acids increased ouabain inhibitable 86Rb+ influx to a level close to that obtained with monensin, indicating that the (Na+,K+)-ATPase is operating near maximal capacity during amino acid transport. This situation may apply to tissue culture conditions and consequently measurements of (Na+,K+)-ATPase activity in buffer solutions alone may yield little information about cation pumping under culture conditions. This finding applies especially to cells having high rates of amino acid transport. Furthermore, rates of amino acid transport may be directly or indirectly involved in the long-term regulation of the number of (Na+,K+)-ATPase molecules in the plasma membrane.     

Effect of salinity on expression of branchial ion transporters in striped bass (Morone saxatilis)

The time course of osmoregulatory adjustments and expressional changes of three key ion transporters in the gill were investigated in the striped bass during salinity acclimations. In three experiments, fish were transferred from fresh water (FW) to seawater (SW), from SW to FW, and from 15-ppt brackish water (BW) to either FW or SW, respectively. Each transfer induced minor deflections in serum [Na+] and muscle water content, both being corrected rapidly (24 hr). Transfer from FW to SW increased gill Na+,K+-ATPase activity and Na+,K+,2Cl- co-transporter expression after 3 days. Abundance of Na+,K+-ATPase alpha-subunit mRNA and protein was unchanged. Changes in Na+,K+,2Cl- co-transporter protein were preceded by increased mRNA expression after 24 hr. Expression of V-type H+-ATPase mRNA decreased after 3 days. Transfer from SW to FW induced no change in expression of gill Na+,K+-ATPase. However, Na+,K+,2Cl- co-transporter mRNA and protein levels decreased after 24 hr and 7 days, respectively. Expression of H+-ATPase mRNA increased in response to FW after 7 days. In BW fish transferred to FW and SW, gill Na+,K+-ATPase activity was stimulated by both challenges, suggesting both a hyper- and a hypo-osmoregulatory response of the enzyme. Acclimation of striped bass to SW occurs on a rapid time scale. This seems partly to rely on the relative high abundance of gill Na+,K+-ATPase and Na+,K+,2Cl- co-transporter in FW fish. In a separate study, we found a smaller response to SW in expression of these ion transport proteins in striped bass when compared with the less euryhaline brown trout. In both FW and SW, NEM-sensitive gill H+-ATPase activity was negligible in striped bass and approximately 10-fold higher in brown trout. This suggests that in striped bass Na+-uptake in FW may rely more on a relatively high abundance/activity of Na+,K+-ATPase compared to trout, where H+-ATPase is critical for establishing a thermodynamically favorable gradient for Na+-uptake.     

Functional Significance of the Effects of Neurotransmitters on the Na+,K+-ATPase System

The aim of the present experiments was to study the effects of the neurotransmitters acetylcholine, noradrenaline, 5-hydroxytryptamine, and dopamine on the Na+,K+-ATPase of rat brain synaptosomal fractions. It is shown that dopamine at low concentrations specifically inhibits the Na+,K+-ATPase of synaptic membranes from the brain regions rich in dopaminergic endings, but has no effect on the synaptosomal Na+,K+-ATPase from the other parts of brain. Acetylcholine and noradrenaline have similar specific effects on Na+,K+-ATPase from cholinergic and adrenergic synaptosomes. The Na+,K+-ATPase of synaptic membranes from the different brain regions, characterised by different distributions of cholinergic, adrenergic, and 5-hydroxytryptaminergic endings, show different reactions with neurotransmitters. These data indicate a functional significance of the effects of the neurotransmitters on the synaptosomal Na+,K+-ATPase.     

Increase of Na+ gradient-dependent L-glutamate and L-aspartate transport in high K+ dog erythrocytes associated with high activity of (Na+, K+)-ATPase

As reported previously, some dogs possess red cells characterized by low Na+, high K+ concentrations, and high activity of (Na+, K+)-ATPase, although normal dog red cells contain low K+, high Na+, and lack (Na+, K+)-ATPase. Furthermore, these red cells show increased activities of L-glutamate and L-aspartate transport, resulting in high accumulations of such amino acids in their cells. The present study demonstrated: (i) Na+ gradient-dependent L-glutamate and L-aspartate transport in the high K+ and low K+ red cells were dominated by a saturable component obeying Michaelis-Menten kinetics. Although no difference of the Km values was observed between the high K+ and low K+ cells, the Vmax values for both amino acids' transport in the high K+ cells were about three times those of low ones. (ii) L- and D-aspartate, but not D-glutamate, competitively inhibited L-glutamate transport in both types of the cells. (iii) Ouabain decreased the uptake of the amino acids in the high K+ dog red cells, whereas it was not effective on those in the low K+ cells. (iv) The ATP-treated high K+ cells [(K+]i not equal to [K+]o, [Na+]i greater than [Na+]o) showed a marked decrease of both amino acids' uptake rate, which was almost the same as that of the low K+ cells. (v) Valinomycin stimulated the amino acids' transport in both of the high K+ and the ATP-treated low K+ cells [( K+]i greater than [K+]o, [Na+]o), suggesting that the transport system of L-glutamate and L-aspartate in both types of the cells might be electrogenic. These results indicate that the increased transport activity in the high K+ dog red cells was a secondary consequence of the Na+ concentration gradient created by (Na+, K+)-ATPase.     

Nantenine and papaverine differentially modify synaptosomal membrane enzymes.

Papaverine (1-[(3,4-Dimethoxyphenyl) methyl]-6,7-dimethoxyisoquinoline) and nantenine (O-methyldomesticine) are chemically related isoquinoline alkaloids displaying similar dose-dependent sedative or convulsant effects, but seem to act differentially on synaptosomal membrane enzymes. Na+, K+-, Mg2+- and Ca2+-ATPase activities were inhibited by nantenine but not by papaverine, whereas acetylcholinesterase activity remained unchanged by nantenine but slightly enhanced by papaverine. Nantenine inhibited roughly both 20-50% Ca2+- and Mg2+-ATPase activities but 40-90% Na+, K+-ATPase activity. Kinetic analysis indicated that nantenine interacts with the substrate ATP for Ca2+-ATPase activity but that it competes with K+ for Na+, K+-ATPase activity. Given the roles of Na+, K+-ATPase and Ca2+-ATPase in cation transport and [Ca2+]i regulation, respectively, the inhibitory effect of nantenine upon these enzymes may explain its convulsant effect though not its sedative activity. The sedative action of both nantenine and papaverine is hardly attributable to an effect on the synaptosomal membrane enzymes assayed.     

Regulation of Na+-K+-ATPase: effect of Mg and Ca ions

The participation of Mg2+ and Ca2+ in complicated mechanisms of Na+, K(+)-ATPase regulation is discussed in the survey. The regulatory actions of Mg2+ on Na+, K(+)-ATPase such as its participation in phosphorylation and dephosphorylation of the enzyme, ADP/ATP-exchange inhibition, cardiac glycosides and vanadate binding with the enzyme, conformational changes induction during ATPase cycle are reviewed in detail. Some current views of mechanisms of above mentioned Mg2+ regulatory effects are discussed. The experimental evidence of Ca2+ immediate influence on the functional activity of Na+, K(+)-ATPase (catalytic, transport and glycoside-binding) are given. It's noted that these effects are based on the conformational changes in the enzyme and also on the phase transition in membrane induced by Ca2+. Unimmediate action of Ca2+ on Na+, K(+)-ATPase is also discussed, especially due to its effect on other membrane systems functionally linked with Na(+)-pump (for instance, due to Na+/Ca(+)-exchanger activation). It's concluded that Mg2+ and Ca2+ as "universal regulators" of the cell effectively influence the functional activity and conformational states of Na+, K(+)-ATPase.     

Kinetic properties of Na+, K+ activated, Mg2+ -dependent ATP-hydrolysis of blood lymphocytes in patients with rheumatoid arthritis and ankylosing spondyloarthritis

The comparative analysis of the kinetic properties of ouabain-sensitive Na+, K+ -ATPase activity of saponin-perforated blood lymphocytes of donors and patients with rheumatoid arthritis (RA) and ankylosing spondyloarthritis (AS) was carried out. When analyzing the alterations in hydrolase activity of the examined enzyme it was shown that in the blood lymphocytes of patients with RA and AS the primary active transport of Na+ and K+ ions is less intensive in comparison with practically healthy donors, but it is characterized by almost the same capacity as in donors. The affinity constant of Na+, K+ -ATPase for ATP in the blood lymphocytes in patients with RA and AS is greater 3.1 and 2.5 times, respectively, in comparison with healthy donor. It was found that in conditions of rheumatic pathology in immunocompetent cells the inhibition of Na+, K+ -ATPase activity is not related to the reduction of maximum reaction rate, but is related to the decrease of Na+, K+ -ATPase affinity to ATP. However, Mg2+ -binding center of Na+, K+ -ATPase in patients with RA and AS remains native. It was identified that the affinity constant of Na+, K+ -ATPase to Na+ ions in the blood lymphocytes of patients with RA and AS is 2.75 times lower than its value in healthy donors. Na+, K+ -ATPase of the blood lymphocytes of patients with RA and AS retains its native receptor properties and sensitivity to ouabain does not change.