Surface Charges of the Membrane Crucially Affect Regulation of Na,K-ATPase by Phospholemman (FXYD1)

The human α₁/His₁₀-β₁ isoform of Na,K-ATPase has been reconstituted as a complex with and without FXYD1 into proteoliposomes of various lipid compositions in order to study the effect of the regulatory subunit on the half-saturating Na⁺ concentration (K _1/2) of Na⁺ ions for activation of the ion pump. It has been shown that the fraction of negatively charged lipid in the bilayer crucially affects the regulatory properties. At low concentrations of the negatively charged lipid DOPS (<10 %), FXYD1 increases K _1/2 of Na⁺ ions for activation of the ion pump. Phosphorylation of FXYD1 by protein kinase A at Ser68 abrogates this effect. Conversely, for proteoliposomes made with high concentrations of DOPS (>10 %), little or no effect of FXYD1 on the K _1/2 of Na⁺ ions is observed. Depending on ionic strength and lipid composition of the proteoliposomes, FXYD1 can alter the K _1/2 of Na⁺ ions by up to twofold. We propose possible molecular mechanisms to explain the regulatory effects of FXYD1 and the influence of charged lipid and protein phosphorylation. In particular, the positively charged C-terminal helix of FXYD1 appears to be highly mobile and may interact with the cytoplasmic N domain of the α-subunit, the interaction being strongly affected by phosphorylation at Ser68 and the surface charge of the membrane.     

Phospholemman phosphorylation alters its fluorescence resonance energy transfer with the Na/K-ATPase pump

Phospholemman (PLM) or FXYD1 is a major cardiac myocyte phosphorylation target upon adrenergic stimulation. Prior immunoprecipitation and functional studies suggest that phospholemman associates with the Na/K-pump (NKA) and mediates adrenergic Na/K-pump regulation. Here, we tested whether the NKA-PLM interaction is close enough to allow fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent (CFP/YFP) fusion proteins of Na/K pump and phospholemman and whether phospholemman phosphorylation alters such FRET. Co-expressed NKA-CFP and PLM-YFP in HEK293 cells co-localized in the plasma membrane and exhibited robust FRET. Selective acceptor photobleach increased donor fluorescence (F(CFP)) by 21.5 +/- 4.1% (n = 13), an effect nearly abolished when co-expressing excess phospholemman lacking YFP. Activation of protein kinase C or A progressively and reversibly decreased FRET assessed by either the fluorescence ratio (F(YFP)/F(CFP)) or the enhancement of donor fluorescence after acceptor bleach. After protein kinase C activation, forskolin did not further reduce FRET, but after forskolin pretreatment, protein kinase C could still reduce FRET. This agreed with phospholemman phosphorylation measurements: by protein kinase C at both Ser-63 and Ser-68, but by protein kinase A only at Ser-68. Expression of PLM-YFP and PLM-CFP resulted in even stronger FRET than for NKA-PLM (F(CFP) increased by 37 +/- 1% upon YFP photobleach), and this FRET was enhanced by phospholemman phosphorylation, consistent with phospholemman multimerization. Co-expressed PLM-CFP and Na/Ca exchange-YFP were highly membrane co-localized, but FRET was undetectable. We conclude that phospholemman and Na/K-pump are in very close proximity (FRET occurs) and that phospholemman phosphorylation alters the interaction of Na/K-pump and phospholemman.     

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.     

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.     

Na,K-ATPase: Isoform structure,function, and expression

An interesting feature of the Na,K-ATPase is the multiplicity of and isoforms. Three isoforms exist for the subunit, 1, 2, and 3, as well for the subunit, 1, 2, and 3. The functional significance of these isoforms is unknown, but they are expressed in a tissue- and developmental-specific manner. For example, all three isoforms of the subunit are present in the brain, while only 1 is present in kidney and lung, and 2 represents the major isoform in skeletal muscle. Therefore, it is possible that each of these isoforms confers different properties on the Na,K-ATPase which allows effective coupling to the physiological process for which it provides energy in the form of an ion gradient. It is also possible that the multiple isoforms are the result of gene triplication and that each isoform exhibits similar enzymatic properties. In this case, the expression of the triplicated genes would be individually regulated to provide the appropriate amount of Na,K-ATPase to the particular tissue and at specific times of development. While differences are observed in such parameters as Na⁺ affinity and sensitivity to cardiac glycosides, it is not known if these properties play a functional role within the cell.Site-directed mutagenesis has identified amino acid residues in the first extracellular region of the subunit as major determinants in the differential sensitivity to cardiac glycosides. Similar studies have failed to identify residues in the second extracellular region involved in cardiac glycoside inhibition. Further analysis of the enzymatic properties of the enzyme, understanding the regulated expression of the genes, and structure-function studies utilizing site-directed mutagenesis should provide new insights into the enzymatic and physiological roles of the various subunit isoforms of the Na,K-ATPase.     

Regulation of Na,K-ATPase by synaptosomal factors and neurotransmitters

The identical increase of Na, K-ATPase activity is caused by oxidated and reduced forms of noradrenaline, serotonin and dopamine through the synaptosomal activating factors. The synaptosomal inhibiting factor, orthovanadate and calcium ions independently inhibit Na, K-ATPase activity. The inhibition constant (Ki) for vanadate does not change in the presence of EDTA, whereas in the presence of synaptosomal factors regulating the Na, K-ATPase factors, noradrenaline causes drastic increase of Ki for vanadate. It has been concluded, that the data point to the existence of special regulating system of brain synaptosomal Na, K-ATPase.     

Regulation of Na, K-ATPase activity by monovalent cations]

A deviation from optimal conditions of the Na, K-ATPase reaction results in a drastic change in the plot: enzyme activity versus Na/K ratio. Acidification of the medium and a decrease in Mg2+ concentration and temperature results in two peaks on the curve at Na/K ratio of about 1 and at Na/K ratio greater than 4. The enhancement of pH of the medium and increase in Mg2+ concentration decreases the first peak and increases the second one. A comparison of these curves for hydrolysis of ATP, UTP and p-nitrophenylphosphate and temperature dependence of the hydrolysis of the substrates suggest that the anomalies observed may be accounted for the Na+ effect on the K-sites or K+ effect on the Na-sites under conditions when cation-binding sites are heterogeneous.     

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.     

Regulation of Na,K-ATPase function in the lens

The two cell types in the lens, epithelium and fiber, have a very different specific activity of Na,K-ATPase; activity is much higher in the epithelium. However, judged by Western blot, fibers and epithelium express a similar amount of both Na,K-ATPase alpha and beta subunit proteins. Na,K-ATPase protein abundance does not tally with Na,K-ATPase activity. Studies were conducted to examine whether protein synthesis plays a role in maintenance of the high Na,K-ATPase activity in lens epithelium. An increase of cytoplasmic sodium was found to increase Na,K-ATPase protein expression in the epithelium, but not in the fibers. The findings illustrate the ability of lens epithelium to synthesize new Na,K-ATPase protein as a way to boost Na,K-ATPase in response to cell damage or pathological events. Methionine incorporation studies suggested Na,K-ATPase synthesis may also play a role in day to day preservation of high Na,K-ATPase activity. Na,K-ATPase protein in lens epithelial cells appeared to be continually synthesized and degraded. Experiments with cycloheximide suggest that specific activity of Na,K-ATPase in the lens epithelium may depend on the ability of the cells to continuously synthesize fresh Na,K-ATPase proteins. However, other factors such as phosphorylation of Na,K-ATPase alpha subunit may also influence Na,K-ATPase activity. When intact lenses were exposed to the agonist thrombin, Na,K-ATPase activity was diminished, but the response was suppressed by inhibitors of the Src family of non-receptor tyrosine kinases. Thrombin elicited tyrosine phosphorylation of lens epithelium membrane proteins, including a 100 kDa protein band thought to be the Na,K-ATPase alpha 1 subunit. It remains to be determined whether a tyrosine phosphorylation mechanism contributes to the low activity of Na,K-ATPase in lens fibers.     

Regulation of Na,K-ATPase Transport Activity by Protein Kinase C

Considerable evidence indicates that the renal Na⁺,K⁺-ATPase is regulated through phosphorylation/dephosphorylation reactions by kinases and phosphatases stimulated by hormones and second messengers. Recently, it has been reported that amino acids close to the NH₂-terminal end of the Na⁺,K⁺-ATPase α-subunit are phosphorylated by protein kinase C (PKC) without apparent effect of this phosphorylation on Na⁺,K⁺-ATPase activity. To determine whether the α-subunit NH₂-terminus is involved in the regulation of Na⁺,K⁺-ATPase activity by PKC, we have expressed the wild-type rodent Na⁺,K⁺-ATPase α-subunit and a mutant of this protein that lacks the first thirty-one amino acids at the NH₂-terminal end in opossum kidney (OK) cells. Transfected cells expressed the ouabain-resistant phenotype characteristic of rodent kidney cells. The presence of the α-subunit NH₂-terminal segment was not necessary to express the maximal Na⁺,K⁺-ATPase activity in cell membranes, and the sensitivity to ouabain and level of ouabain-sensitive Rb⁺-transport in intact cells were the same in cells transfected with the wild-type rodent α1 and the NH₂-deletion mutant cDNAs. Activation of PKC by phorbol 12-myristate 13-acetate increased the Na⁺,K⁺-ATPase mediated Rb⁺-uptake and reduced the intracellular Na⁺ concentration of cells transfected with wild-type α1 cDNA. In contrast, these effects were not observed in cells expressing the NH₂-deletion mutant of the α-subunit. Treatment with phorbol ester appears to affect specifically the Na⁺,K⁺-ATPase activity and no evidence was observed that other proteins involved in Na⁺-transport were affected. These results indicate that amino acid(s) located at the α-subunit NH₂-terminus participate in the regulation of the Na⁺,K⁺-ATPase activity by PKC. Received: 10 July 1996/Revised: 19 September 1996     

Molecular Mechanisms of the Redox Regulation of the Na,K-ATPase

Biophysics - This review considers the molecular mechanisms involved in the redox regulation of the Na,K-ATPase. The enzyme creates a transmembrane gradient of sodium and potassium ions, which is...     

Regulation of Na,K-ATPase during acute lung injury

A hallmark of acute lung injury is the accumulation of a protein rich edema which impairs gas exchange and leads to hypoxemia. The resolution of lung edema is effected by active sodium transport, mostly contributed by apical Na⁺ channels and the basolateral located Na,K-ATPase. It has been reported that the decrease of Na,K-ATPase function seen during lung injury is due to its endocytosis from the cell plasma membrane into intracellular pools. In alveolar epithelial cells exposed to severe hypoxia, we have reported that increased production of mitochondrial reactive oxygen species leads to Na,K-ATPase endocytosis and degradation. We found that this regulated process follows what is referred as the Phosphorylation–Ubiquitination–Recognition–Endocytosis–Degradation (PURED) pathway. Cells exposed to hypoxia generate reactive oxygen species which activate PKCζ which in turn phosphorylates the Na,K-ATPase at the Ser18 residue in the N-terminus of the α1-subunit leading the ubiquitination of any of the four lysines (K16, K17, K19, K20) adjacent to the Ser18 residue. This process promotes the α1-subunit recognition by the μ2 subunit of the adaptor protein-2 and its endocytosis trough a clathrin dependent mechanism. Finally, the ubiquitinated Na,K-ATPase undergoes degradation via a lysosome/proteasome dependent mechanism.     

Regulation of the number of k-binding sites of Na,K-ATPase by adenosine triphosphate

A possible existence of two functional states of Na,K-ATPase with different electrogenic coefficients has been experimentally proved. Regulation of electrogenicity is achieved by alteration in the number of K+ transport sites. A transition of Na,K-ATPase from one functional state to the other has been shown to occur during the binding of ATP free ions.     

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.     

Regulation of inositol 1,4,5-trisphosphate receptor-mediated calcium release by the Na/K-ATPase in cultured renal epithelial cells

It is known that the Na/K-ATPase alpha1 subunit interacts directly with inositol 1,4,5-triphosphate (IP(3)) receptors. In this study we tested whether this interaction is required for extracellular stimuli to efficiently regulate endoplasmic reticulum (ER) Ca(2+) release. Using cultured pig kidney LLC-PK1 cells as a model, we demonstrated that graded knockdown of the cellular Na/K-ATPase alpha1 subunit resulted in a parallel attenuation of ATP-induced ER Ca(2+) release. When the knockdown cells were rescued by knocking in a rat alpha1, the expression of rat alpha1 restored not only the cellular Na/K-ATPase but also ATP-induced ER Ca(2+) release. Mechanistically, this defect in ATP-induced ER Ca(2+) release was neither due to the changes in the amount or the function of cellular IP(3) and P2Y receptors nor the ER Ca(2+) content. However, the alpha1 knockdown did redistribute cellular IP(3) receptors. The pool of IP(3) receptors that resided close to the plasma membrane was abolished. Because changes in the plasma membrane proximity could reduce the efficiency of signal transmission from P2Y receptors to the ER, we further determined the dose-dependent effects of ATP on protein kinase Cepsilon activation and ER Ca(2+) release. The data showed that the alpha1 knockdown de-sensitized the ATP-induced ER Ca(2+) release but not PKCepsilon activation. Moreover, expression of the N terminus of Na/K-ATPase alpha1 subunit not only disrupted the formation of the Na/K-ATPase-IP(3) receptor complex but also abolished the ATP-induced Ca(2+) release. Finally, we observed that the alpha1 knockdown was also effective in attenuating ER Ca(2+) release provoked by angiotensin II and epidermal growth factor.     

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 Na,K-ATPase biosynthesis in developing Artemia salina

Regulation of the biosynthesis of the sodium- and potassium-activated adenosine triphosphatase (Na,K-ATPase) (EC 3.6.1.3) was studied in the developing brine shrimp, Artemia salina. Measurement of levels of the subunits of the Na,K-ATPase by radioimmunoassay indicated the presence of both alpha and beta subunits in undeveloped cysts and developing embryos prior to the appearance of enzymatic activity. The quantity of each subunit increased dramatically between 8 and 24 h of development and then reached a plateau at about 32 h. The quantities of translationally active mRNA alpha and mRNA beta were also determined. Undeveloped cysts contained mRNA alpha and mRNA beta, and the amounts increased 9- and 3-fold, respectively, during the first 24 h of development. The data suggest that the increase in Na,K-ATPase activity was at least in part due to increases in protein synthesis related to changes in mRNA levels. The data also suggest involvement of additional regulatory mechanisms. The alpha-subunit has been detected as two molecular weight forms (alpha 1 and alpha 2) which demonstrate changes in relative amounts during development (Peterson, G. L., Churchill, L., Fisher, J. A., and Hokin, L. E. (1982) J. Exp. Zool. 221, 295-308). We show here that this was not due to changes in mRNA alpha 1 and mRNA alpha 2.     

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.     

Purification of the human alpha2 Isoform of Na,K-ATPase expressed in Pichia pastoris. Stabilization by lipids and FXYD1

Human alpha1 and alpha2 isoforms of Na,K-ATPase have been expressed with porcine 10*Histidine-tagged beta1 subunit in Pichia pastoris. Methanol-induced expression of alpha2 is optimal at 20 degrees C, whereas at 25 degrees C, which is optimal for expression of alpha1, alpha2 is not expressed. Detergent-soluble alpha2beta1 and alpha1beta1 complexes have been purified in a stable and functional state. alpha2beta1 shows a somewhat lower Na,K-ATPase activity and higher K0.5K compared to alpha1beta1, while values of K0.5Na and KmATP are similar. Ouabain inhibits both alpha1beta1 (K0.5 24.6 +/- 6 nM) and alpha2beta1 (K0.5 102 +/- 14 nM) with high affinity. A striking difference between the isoforms is that alpha2beta1 is unstable. Both alpha1beta1 and alpha2beta1 complexes, prepared in C12E8 with an added phosphatidyl serine, are active, but alpha2beta1 is rapidly inactivated at 0 degrees C. Addition of low concentrations of cholesterol with 1-stearoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (SOPS) stabilizes strongly, maintaining alpha2beta1 active up to two weeks at 0 degrees C. By contrast, alpha1beta1 is stable at 0 degrees C without added cholesterol. Both alpha1beta1 and alpha2beta1 complexes are stabilized by cholesterol at 37 degrees C. Human FXYD1 spontaneously associates in vitro with either alpha1beta1 or alpha2beta1, to form alpha1beta1/FXYD1 and alpha2beta1/FXYD1 complexes. The reconstituted FXYD1 protects both alpha1beta1 and alpha2beta1 very strongly against thermal inactivation. Instability of alpha2 is attributable to suboptimal phophatidylserine-protein interactions. Residues within TM8, TM9 and TM10, near the alphabeta subunit interface, may play an important role in differential interactions of lipid with alpha1 and alpha2, and affect isoform stability. Possible physiological implications of isoform interactions with phospholipids and FXYD1 are discussed.