Oxidative Resistance of Na/K-ATPase

1. Oxidative modification of Na/K-ATPase from brain and kidney has been studied. Brain enzyme has been found to be more sensitive than kidney enzyme to inhibition by both H₂O₂ and NaOCl.2. The inhibition of Na/K-ATPase correlates well with the decrease in a number of SH groups, suggesting that the latter belong mainly to ATPase protein and are essential for the enzyme activity. We suggest that the differences in the number, location, and accessibility of SH groups in Na/K-ATPase isozymes predict their oxidative stability.3. The hydrophilic natural antioxidant carnosine, the hydrophobic natural antioxidant -tocopherol, and the synthetic antioxidant ionol as well as the ferrous ion chelating agent deferoxamine were found to protect Na/K-ATPase from oxidation by different concentrations of H₂O₂. The data suggest that these antioxidants are effective due to their ability to neutralize or to prevent formation of hydroxyl radicals.     

Na/K-ATPase as an oligomeric ensemble

Differences in the kinetic behavior and properties of monomeric and oligomeric forms of membrane-bound Na/K-ATPase are analyzed. It is concluded that enzyme molecules within oligomeric complexes are affected by extrinsic signals that result in change of enzyme activity, whereas the individual (protomeric) state is insensitive to these signals. Some of the major factors of such regulation are microviscosity of the lipid environment, reactive oxygen species, and intracellular protein kinases.     

Terahertz vibration modes in Na/K-ATPase

Mechanical vibration in the Terahertz range is believed to be connected with protein functions. In this paper, we present the results of a normal-mode analysis (modal analysis) of a Na/K-ATPase all-atom model, focusing the attention on low-frequency vibration modes. The numerical model helps in the interpretation of experimental results previously obtained by the authors via Raman spectroscopy of Na/K-ATPase samples, where several unassigned peaks were found in the sub-500 cm^?1 range. In particular, vibration modes corresponding to peaks at 27, 190 and 300 cm^?1, found experimentally, are confirmed here numerically, together with some other modes at lower frequencies (wavenumbers) that were not possible to observe in the experimental test. All the aforementioned modes correspond to vibrations involving the protein ends, i.e. portions directly related to the operating mechanism of the sodium-potassium pump.     

The anticancer drug perillyl alcohol is a Na/K-ATPase inhibitor

Indoleamine 2,3-dioxygenase (IDO) is an enzyme that suppresses adaptive T-cell immunity by catabolizing tryptophan from the cellular microenvironment. Inhibition of IDO pathway might enhance the efficacy of immunotherapeutic strategies for cancer. We synthesized 1-alkyl-tryptophan targeted IDO inhibitors and compared their effects on IDO expression and activity in dendritic cells (DCs) with the common IDO inhibitor 1-methyl-dl-tryptophan (1-MT). The IDO gene expression was examined by RT-PCR and realtime PCR. The toxicity of these analogs on the proliferation of DCs was detected by MTT assay. All of these analogs inhibited IDO expression and activity induced by IFN-γ and showed no cytotoxicity to DCs at 100 μM. 1-MT intensively suppressed IDO1 expression and activity in DCs, and 1-propyl-tryptophan (1-PT) and 1-isopropyl-tryptophan (1-isoPT) moderately inhibited them. 1-Butyl-tryptophan (1-BT) and 1-ethyl-tryptophan (1-ET) mainly inhibited IDO2 expression. Our results suggest that those analogs differed in their inhibitory activity on IDO expression may give us a clue for developing active IDO inhibitors.     

The Na,K-ATPase

The energy dependent exchange of cytoplasmic Na⁺ for extracellular K⁺ in mammalian cells is due to a membrane bound enzyme system, the Na,K-ATPase. The exchange sustains a gradient for Na⁺ into and for K⁺ out of the cell, and this is used as an energy source for creation of the membrane potential, for its de- and repolarisation, for regulation of cytoplasmic ionic composition and for transepithelial transport. The Na,K-ATPase consists of two membrane spanning polypeptides, an -subunit of 112-kD and a -subunit, which is a glycoprotein of 35-kD. The catalytic properties are associated with the -subunit, which has the binding domain for ATP and the cations. In the review, attention will be given to the biochemical characterization of the reaction mechanism underlying the coupling between hydrolysis of the substate ATP and transport of Na⁺ and K⁺.     

Identification of the 5-iodoacetamidofluorescein reporter site on the Na,K-ATPase

5-Iodoacetamidofluorescein (5-IAF) labels the catalytic (alpha) subunit of dog kidney Na,K-ATPase without inhibiting enzymatic activity and is thus a useful fluorescent reporter of enzyme conformation under conditions of enzyme turnover. In this study conditions for labeling a unique sulfhydryl group are described, and this residue is identified in the cDNA-derived sequence. Reaction with iodoacetate (IAA) prior to fluorescent labeling lowers the stoichiometry of 5-IAF incorporation from 2.1 to 1.2 mol/mol alpha beta protomer, and increases the conformationally dependent fluorescence changes by 40-50%, consistent with the elimination of nonspecific labeling. IAA/IAF-enzyme has the same catalytic activity as the IAF-enzyme. In contrast, treatment with iodoacetamide prior to labeling with 5-IAF abolishes all fluorescence responses, although activity is retained. IAA/IAF-enzyme was digested by extensive trypsinolysis, and the fluorescent peptides released from the membrane were purified by high performance liquid chromatography and sequenced. Several fluorescent peptides were found, containing all or part of the sequence Cys-Ile-Glu-Leu-Cys-Cys-Gly-Ser-Val-Lys, corresponding to residues 452-461 in the sheep alpha subunit. The major site of modification is the second of the vicinal cysteine residues, Cys-457. Phenylarsine oxide, a reagent specific for vicinal sulfhydryl groups, prevents fluorophore incorporation, thereby confirming the identification of the IAF site from the sequence data.     

The Na/K-ATPase regulation by neurotransmitters in ontogeny

Activity of the Na/K-ATPase from rat brain synaptic membranes is inhibited by NA (noradrenaline). However, during fractionation of cytozole from nerve endings, two non-homogeneous peaks are found (SF(a), 60-100 kD and SF( i ),;10 kD), which influence the Na/K-ATPase activity, both directly and SF(a) NA-dependently. Joint action of NA and synaptic factors (SF(a) and SF(i)) on the Na/K-ATPase, represents a sum of four different processes: 1) NA, without synaptic factors, inhibits the Na/K-ATPase; 2) At low SF(a) concentrations NA-dependent Na/K-ATPase activatory mechanism is evident; 3) At high SF(a) concentrations NA-independent Na/K-ATPase is activated; 4) The low-molecular SF(i) protein inhibits the Na/K-ATPase. Regulation of the Na/K-ATPase activity by NA, SF(a) and SF( i), obtained in similar conditions from two weeks old and one year old rats, is different. In older rats SF(i) is characterized with strong Na/K-ATPase inhibition; in younger rats SF(i) does not change the Na/K-ATPase activity. The NA- and SF(i) -dependent inhibition and activation ratio is different in young and elder rats. In two week olds NA/SF(i) activatory mechanism is stronger, while in one year olds NA-dependent inhibition of the Na/K-ATPase is prevailing. These experimental data indicate that regulation of the Na/K-ATPase activity has an important role in synaptic transmission and that this process has noteworthy, albeit presently unknown, functional importance in integrative activity of the brain.     

Regulation of alpha1 Na/K-ATPase expression by cholesterol

We have reported that α1 Na/K-ATPase regulates the trafficking of caveolin-1 and consequently alters cholesterol distribution in the plasma membrane. Here, we report the reciprocal regulation of α1 Na/K-ATPase by cholesterol. Acute exposure of LLC-PK1 cells to methyl β-cyclodextrin led to parallel decreases in cellular cholesterol and the expression of α1 Na/K-ATPase. Cholesterol repletion fully reversed the effect of methyl β-cyclodextrin. Moreover, inhibition of intracellular cholesterol trafficking to the plasma membrane by compound U18666A had the same effect on α1 Na/K-ATPase. Similarly, the expression of α1, but not α2 and α3, Na/K-ATPase was significantly reduced in the target organs of Niemann-Pick type C mice where the intracellular cholesterol trafficking is blocked. Mechanistically, decreases in the plasma membrane cholesterol activated Src kinase and stimulated the endocytosis and degradation of α1 Na/K-ATPase through Src- and ubiquitination-dependent pathways. Thus, the new findings, taken together with what we have already reported, revealed a previously unrecognized feed-forward mechanism by which cells can utilize the Src-dependent interplay among Na/K-ATPase, caveolin-1, and cholesterol to effectively alter the structure and function of the plasma membrane.     

Energetics and the design principles of the Na/K-ATPase

Following on the pioneering analysis by Pickart and Jencks of the energetics of the calcium pump, the present mini review attempts a similar analysis of the somewhat more complicated sodium--and potassium--activated ATPase pump-enzyme. The analysis is based on the measurements of the rate-constants of the individual steps in the enzymic reaction which brings about pumping and uses data assembled by Stürmer et al., in the laboratory of Peter L?uger in Konstanz. The aim of such an analysis is to calculate the overall free energy released on ATP hydrolysis and to apportion this energy among the successive steps of the pump-enzyme reaction. We calculate the so-called basic free energy changes that take into account the prevailing ligand and ion concentrations, rather than the standard-state free energies that refer to 1 M concentrations of these ions and ligands. Using appropriate values of the ion and ligand concentrations in cardiac muscle, the available free energy which can be released from the hydrolysis of ATP (at 20 degrees C) comes out at 575 mV. Following a complete cycle of pumping, 371 mV of this free energy are found stored in the sodium and potassium ion gradients. The remaining 204 mV from the free energy of hydrolysis of ATP are lost to the ATP system. This part of the energy, that had been transduced into the concentration gradient of sodium, has presumably been used in the living cell to drive the co- and counter-transport (symport and antiport) of ions and metabolites in secondary transports. The free energy changes are pretty evenly apportioned along the various steps in the pumping cycle. The steps that one might naively have thought to be "powered", such as the step in which covalently bound phosphate is transformed from a high-energy to a low-energy state, or the step in which sodium is released into the phase containing a high concentration of sodium, show some of the lowest drops of free energy, 61 mV and 27 mV, respectively. The most surprising step in the overall reaction of ATP hydrolysis and synthesis is the phosphorylation of the protein from inorganic phosphate with formation of the acylphosphate bond. The stabilization of the acylphosphate bond presumably arises from ionic interactions between the covalently bound phosphate itself and appropriate groupings on the enzyme. ATP formation on the F0F1 ATPase (the F-type ATPase) is in an analogous way stabilized in the first place by phospho-ligand/enzyme interactions.     

The genes of Na,K-ATPase,a selfreview

The review is devoted to analysis of research carried out in the author's laboratory on structure-function relationships in genes coding for Na, K-ATPases. Also considered are problems related to molecular evolution of ion-transporting ATPases.This brief review is devoted to a fragment of research carried out in my laboratory, the Laboratory of Human Genes Structure and Function at the Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences. The area of the review may be named as structural-evolutionary analysis of functional anatomies of genes. The approach is fairly standard and its essence was formulated long ago: evolution decides to be or not to be based on usefulness or lack of it. The elements of genes that are important for the gene function are retained in the course of evolution, and a comparison of genes having similar functions in different species should, hopefully, reveal different behavior of gene blocks, conservation of functionally significant blocks and variability of less significant or insignificant ones. An approach like this has been widely used in comparing proteins. However, a study of genes gives the investigator yet another tool of structural and evolutionary import: the exon structure may be relevant to the gene's evolutionary history, with exons corresponding to the functional domains arguments for and against this fascinating hypothesis have been reviewed by Blake (Blake, 1985).However, even if the exon-domain correlation does not hold in the general case, a similarity in the exon-intron pattern of genes from different species is indicative of their common evolutionary origin and is enforcing the logic of variability analysis, provided, of course, that the compared genes have a common predecessor.A few years ago we employed this approach to analyze the functional structure of genes coding for subunits of bacterial DNA-dependent RNA polymerases and constructed functional maps of the enzyme. After that, a similar study of Na,K-ATPase genes to be reviewed here was started. The entire project became possible through collaboration with the lab of Dr. N. N. Modyanov, an eminent specialist in protein chemistry who had already accumulated considerable information on Na,K-ATPase from pig kidneys by that time. I would also like to stress that the work has been started on the initiative of the deceased Director of the Institute, Yu. A. Ovchinnikov. Since this is a self-review, I am asking my colleagues whose work will not be cited here to excuse me. More objective information can be found in the reviews published on the subject (Cantley, 1981, Jorgensen et al., 1982; Lingrel et al., 1989; Pedersen & Carafoli, 1987; Rossier et al., 1987; Sweadner, 1989).     

Phosphorylation of the Na,K-ATPase and the H,K-ATPase

Phosphorylation is a widely used, reversible means of regulating enzymatic activity. Among the important phosphorylation targets are the Na⁺,K⁺- and H⁺,K⁺-ATPases that pump ions against their chemical gradients to uphold ionic concentration differences over the plasma membrane. The two pumps are very homologous, and at least one of the phosphorylation sites is conserved, namely a cAMP activated protein kinase (PKA) site, which is important for regulating pumping activity, either by changing the cellular distribution of the ATPases or by directly altering the kinetic properties as supported by electrophysiological results presented here. We further review the other proposed pump phosphorylations.     

Low-angle neutron scattering analysis of Na/K-ATPase in detergent solution

Purified Na/K-ATPase from guinea pig renal outer medulla has been delipidated and solubilized in Brij 58 (polyoxyethylene ether; C-16, E-20). At a concentration of 2 mg of Brij 58/mg of protein, about one-half the enzyme complement was solubilized and almost 50% of Na/K-ATPase activity was retained by the enzyme-micelle complex. Guinier plots of the neutron scattering profiles yielded no evidence of heterogeneity with respect to subunit composition or the state of aggregation in the solubilized oligomers. Contrast matching with D2O used to obtain estimates of the molecular weight of the micellar form of Na/K-ATPase gave a mean value of 310,000 +/- 42,700, which corresponds to an alpha 2 beta 2 tetramer. A Stuhrmann plot of the neutron scattering data yielded an estimated radius of gyration of 67 A. The Stuhrmann plot also indicated an asymmetrical distribution of neutron scattering density. On the basis of the Stuhrmann plot parameters, the estimated molecular weight, and the radius of gyration, a low-resolution model was formulated of the oligomeric unit of Na/K-ATPase.     

Regulation of caveolin-1 membrane trafficking by the Na/K-ATPase

Here, we show that the Na/K-ATPase interacts with caveolin-1 (Cav1) and regulates Cav1 trafficking. Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface. These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase α1 subunit. Moreover, knockdown of the Na/K-ATPase increases basal levels of active Src and stimulates endocytosis of Cav1 from the plasma membrane. Microtubule-dependent long-range directional trafficking in Na/K-ATPase–depleted cells results in perinuclear accumulation of Cav1-positive vesicles. Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi. Thus, the Na/K-ATPase regulates Cav1 endocytic trafficking and stabilizes the Cav1 plasma membrane pool.     

Identification of the cofilin-binding sites in the large cytoplasmic domain of Na,K-ATPase

Na,K-ATPase, an alpha, beta heterodimer, is found in the plasma membrane of all animal cells. The alpha chain is believed to have 10 transmembrane regions and a large cytoplasmic domain between the 4th and 5th transmembrane regions (H4-H5). In our previous report, the large (3rd) cytoplasmic domains of the alpha1 and alpha2 isoform were found to interact with cofilin, an actin-modulating protein, by the yeast two-hybrid system. Here we show that cofilin interacts only with the 3rd cytoplasmic domain of the alpha2 subunit but not with the 2nd, 4th, and 5th cytoplasmic domains or the cytoplasmic region of the beta subunit of Na,K-ATPase. We also demonstrate that cofilin interacts with the large cytoplasmic domains of the alpha1, alpha2 and alpha3 isoforms of Na,K-ATPase, but not with those of glucose transporter 1, glucose transporter 4, cystic fibrosis transmembrane conductance regulator and plasma membrane Ca-ATPase. We introduced 10 mutations into the 3rd cytoplasmic domain of Na,K-ATPase to identify the binding sites with cofilin. Eight of these mutants were single amino acid substitutions (R417Q, K470Q, K654G, D672A, K691A, R700G, R700A and D710G) and two were double mutant (K654GR700G and K719AK720A). Analysis of the activity of the reporter gene of these mutants shows that residues D672 and R700 of the 3rd cytoplasmic domain of Na,K-ATPase are involved in the interaction with cofilin.     

Functional characterization of Src-interacting Na/K-ATPase using RNA interference assay

We have shown that the Na/K-ATPase and Src form a signaling receptor complex. Here we determined how alterations in the amount and properties of the Na/K-ATPase affect basal Src activity and ouabain-induced signal transduction. Several alpha1 subunit knockdown cell lines were generated by transfecting LLC-PK1 cells with a vector expressing alpha1-specific small interference RNA. Although the alpha1 knockdown resulted in significant decreases in Na/K-ATPase activity, it increased the basal Src activity and tyrosine phosphorylation of focal adhesion kinase, a Src effector. Concomitantly it also abolished ouabain-induced activation of Src and ERK1/2. When the knockdown cells were rescued by a rat alpha1, both Na/K-ATPase activity and the basal Src activity were restored. In addition, ouabain was able to stimulate Src and ERK1/2 in the rescued cells at a much higher concentration, consistent with the established differences in ouabain sensitivity between pig and rat alpha1. Finally both fluorescence resonance energy transfer analysis and co-immunoprecipitation assay indicated that the pumping-null rat alpha1 (D371E) mutant could also bind Src. Expression of this mutant restored the basal Src activity and focal adhesion kinase tyrosine phosphorylation. Taken together, the new findings suggest that LLC-PK1 cells contain a pool of Src-interacting Na/K-ATPase that not only regulates Src activity but also serves as a receptor for ouabain to activate protein kinases.     

Na/K-ATPase under Oxidative Stress: Molecular Mechanisms of Injury

1. The authors compare oxidative injury to brain and kidney Na/K-ATPase using in vitro and in vivo approaches. The substrate dependence of dog kidney Na/K-ATPase was examined both before and after partial hydrogen peroxide modification. A computer simulation model was used for calculating kinetic parameters.2. The substrate dependence curve for the unmodified endogenous enzyme displayed a typical curve with an intermediate plateau, adequately described by the sum of hyperbolic and sigmoidal components.3. The modified enzyme demonstrated a dependent curve that closely approximates normal hyperbola. The estimated ATP K _m value for the endogenous enzyme was about 85 M; the K _h was equal to 800 M. The maximal number of protomers interacting was 8. Following oxidative modification, the enzyme substrate dependence curve did not show a significant change in the maximal protomer rate V _m, while the K _m was increased slightly and interprotomer interaction was abolished.4. Na/K-ATPase from an ischemic gerbil brain showed a 22% decrease in specific activity. The maximal rate of ATP hydrolysis by an enzyme protomer changed slightly, but the sigmoidal component, characterizing the enzyme's ability to form oligomers was abolished completely. The K _m value was almost unchanged, but the Hill coefficient fell to 1. These data show that Na/K-ATPase molecules isolated from the ischemic brain have lost the ability to interact with one another.5. We suggest that the most important consequence of oxidative modification is Na/K-ATPase oligomeric structure formation and subsequent hydrolysis rate suppression.     

Regulation of Intracellular Cholesterol Distribution by Na/K-ATPase

Recent studies have ascribed many non-pumping functions to the Na/K-ATPase. We show here that graded knockdown of cellular Na/K-ATPase α1 subunit produces a parallel decrease in both caveolin-1 and cholesterol in light fractions of LLC-PK1 cell lysates. This observation is further substantiated by imaging analyses, showing redistribution of cholesterol from the plasma membrane to intracellular compartments in the knockdown cells. Moreover, this regulation is confirmed in α1^+/– mouse liver. Functionally, the knockdown-induced redistribution appears to affect the cholesterol sensing in the endoplasmic reticulum, because it activates the sterol regulatory element-binding protein pathway and increases expression of hydroxymethylglutaryl-CoA reductase and low density lipoprotein receptor in the liver. Consistently, we detect a modest increase in hepatic cholesterol as well as a reduction in the plasma cholesterol. Mechanistically, α1^+/– livers show increases in cellular Src and ERK activity and redistribution of caveolin-1. Although activation of Src is not required in Na/K-ATPase-mediated regulation of cholesterol distribution, the interaction between the Na/K-ATPase and caveolin-1 is important for this regulation. Taken together, our new findings demonstrate a novel function of the Na/K-ATPase in control of the plasma membrane cholesterol distribution. Moreover, the data also suggest that the plasma membrane Na/K-ATPase-caveolin-1 interaction may represent an important sensing mechanism by which the cells regulate the sterol regulatory element-binding protein pathway.The Na/K-ATPase, also called the sodium pump, is an ion transporter that mediates active transport of Na⁺ and K⁺ across the plasma membrane by hydrolyzing ATP (1, 2). The functional sodium pump is mainly composed of α and β subunits. The α subunit is the catalytic component of the holoenzyme; it contains both the nucleotide and the cation binding sites (3). So far, four isoforms of α subunit have been discovered, and each one shows a distinct tissue distribution pattern (4, 5). Interestingly, studies during the past few years have uncovered many non-pumping functions of Na/K-ATPase (6–10). Recently, we have demonstrated that more than half of the Na/K-ATPase may actually perform cellular functions other than ion pumping at least in LLC-PK1 cells (11). Moreover, the non-pumping pool of Na/K-ATPase mainly resides in caveolae and interacts with a variety of proteins such as Src, inositol 1,4,5-trisphosphate receptor, and caveolin-1 (12–14). While the interaction between Na/K-ATPase and inositol 1,4,5-trisphosphate receptor facilitates Ca²⁺ signaling (13) the dynamic association between Na/K-ATPase and Src appears to be an essential step for ouabain to stimulate cellular kinases (15). More recently, we report that the interaction between the Na/K-ATPase and caveolin-1 plays an important role for the membrane trafficking of caveolin-1. Knockdown of the Na/K-ATPase leads to altered subcellular distribution of caveolin-1 and increases the mobility of caveolin-1-containing vesicles (16).Caveolin is a protein marker for caveolae (17). Caveolae are flask-shaped vesicular invaginations of plasma membrane and are enriched in cholesterol, glycosphingolipids, and sphingomyelin (18). There are three genes and six isoforms of caveolin. Caveolin-1 is a 22-kDa protein and is expressed in many types of cells, including epithelial and endothelial cells. In addition to their role in biogenesis of caveolae (19), accumulating evidence has implicated caveolin proteins in cellular cholesterol homeostasis (20). For instance, caveolin-1 directly binds to cholesterol in a 1:1 ratio (21). It was also found to be an integral member of the intracellular cholesterol trafficking machinery between internal membranes and plasma membrane (22, 23). The expression of caveolin-1 appears to be under control of SREBPs,² the master regulators of intracellular cholesterol level (24). Furthermore, knockout of caveolin-1 significantly affected cholesterol metabolism in mouse embryonic fibroblasts and mouse peritoneal macrophages (25). Because we found that the Na/K-ATPase regulates cellular distribution of caveolin-1, we propose that it may also affect intracellular cholesterol distribution and metabolism. To test our hypothesis, we have investigated whether sodium pump α1 knockdown affects cholesterol distribution and metabolism both in vitro and in vivo. Our results indicate that sodium pump α1 expression level plays a role in the proper distribution of intracellular cholesterol. Down-regulation of sodium pump α1 not only redistributes cholesterol between the plasma membrane and cytosolic compartments, but also alters cholesterol metabolism in mice.     

Na/K-ATPase tethers phospholipase C and IP3 receptor into a calcium-regulatory complex

We have shown that the caveolar Na/K-ATPase transmits ouabain signals via multiple signalplexes. To obtain the information on the composition of such complexes, we separated the Na/K-ATPase from the outer medulla of rat kidney into two different fractions by detergent treatment and density gradient centrifugation. Analysis of the light fraction indicated that both PLC-gamma1 and IP3 receptors (isoforms 2 and 3, IP3R2 and IP3R3) were coenriched with the Na/K-ATPase, caveolin-1 and Src. GST pulldown assays revealed that the central loop of the Na/K-ATPase alpha1 subunit interacts with PLC-gamma1, whereas the N-terminus binds IP3R2 and IP3R3, suggesting that the signaling Na/K-ATPase may tether PLC-gamma1 and IP3 receptors together to form a Ca(2+)-regulatory complex. This notion is supported by the following findings. First, both PLC-gamma1 and IP3R2 coimmunoprecipitated with the Na/K-ATPase and ouabain increased this interaction in a dose- and time-dependent manner in LLC-PK1 cells. Depletion of cholesterol abolished the effects of ouabain on this interaction. Second, ouabain induced phosphorylation of PLC-gamma1 at Tyr(783) and activated PLC-gamma1 in a Src-dependent manner, resulting in increased hydrolysis of PIP2. It also stimulated Src-dependent tyrosine phosphorylation of the IP3R2. Finally, ouabain induced Ca(2+) release from the intracellular stores via the activation of IP3 receptors in LLC-PK1 cells. This effect required the ouabain-induced activation of PLC-gamma1. Inhibition of Src or depletion of cholesterol also abolished the effect of ouabain on intracellular Ca(2+).     

Active transport of Na+ by modified Na,K-ATPase

The ability of ATP, CTP, ITP, GTP and UTP to induce ouabain-sensitive accumulation of Na+ by proteoliposomes with a reconstituted Na/K-pump was studied. At low Na+/K+ ratio (20 mM/50 mM), a correlation was observed between the proton-accepting capacity of the nucleotide and its efficiency as an active transport substrate. In order to test the hypothesis on the role of the negative charge in position 1 of the purine (3-pyrimidine) base of the nucleotide in the reversible transitions from the Na- to the K-conformations of Na,K-ATPase, two ATP analogs (N1-hydroxy-ATP possessing a proton-accepting ability and N1-methoxy-ATP whose molecule carries a negative charge quenched by a methyl group) were used. The first substrate provides for active accumulation of Na+ by proteoliposomes at a rate similar to that of ATP, whereas the second substrate is fairly ineffective.