Structural Insights into the Interactions of Digoxin and Na+/K+-ATPase and Other Targets for the Inhibition of Cancer Cell Proliferation.

Digoxin is a cardiac glycoside long used to treat congestive heart failure and found recently to show antitumor potential. The hydroxy groups connected at the C-12, C-14, and C-3'a positions; the C-17 unsaturated lactone unit; the conformation of the steroid core; and the C-3 saccharide moiety have been demonstrated as being important for digoxin's cytotoxicity and interactions with Na+/K+-ATPase. The docking profiles for digoxin and several derivatives and Na+/K+-ATPase were investigated; an additional small Asn130 side pocket was revealed, which could be useful in the design of novel digoxin-like antitumor agents. In addition, the docking scores for digoxin and its derivatives were found to correlate with their cytotoxicity, indicating a potential use of these values in the prediction of the cancer cell cytotoxicity of other cardiac glycosides. Moreover, in these docking studies, digoxin was found to bind to FIH-1 and NF-κB but not HDAC, IAP, and PI3K, suggesting that this cardiac glycoside directly targets FIH-1, Na+/K+-ATPase, and NF-κB to mediate its antitumor potential. Differentially, digoxigenin, the aglycon of digoxin, binds to HDAC and PI3K, but not FIH-1, IAP, Na+/K+-ATPase, and NF-κB, indicating that this compound may target tumor autophagy and metabolism to mediate its antitumor propensity.

Membrane accessibility of glutathione.

Regulation of the ion pumping activity of the Na+,K+-ATPase is crucial to the survival of animal cells. Recent evidence has suggested that the activity of the enzyme could be controlled by glutathionylation of cysteine residue 45 of the ß-subunit. Crystal structures so far available indicate that this cysteine is in a transmembrane domain of the protein. Here we have analysed via fluorescence and NMR spectroscopy as well as molecular dynamics simulations whether glutathione is able to penetrate into the interior of a lipid membrane. No evidence for any penetration of glutathione into the membrane was found. Therefore, the most likely mechanism whereby the cysteine residue could become glutathionylated is via a loosening of the α-ß subunit association, creating a hydrophilic passageway between them to allow access of glutathione to the cysteine residue. By such a mechanism, glutathionylation of the protein would be expected to anchor the modified cysteine residue in a hydrophilic environment, inhibiting further motion of the ß-subunit during the enzyme's catalytic cycle and suppressing enzymatic activity, as has been experimentally observed. The results obtained, therefore, suggest a possible structural mechanism of how the Na+,K+-ATPase could be regulated by glutathione.

Stabilisation of Na,K-ATPase structure by the cardiotonic steroid ouabain.

Cardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism spectroscopy we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography.

Spatial distribution of Na+-K+-ATPase in dendritic spines dissected by nanoscale superresolution STED microscopy.

BACKGROUND: The Na+,K+-ATPase plays an important role for ion homeostasis in virtually all mammalian cells, including neurons. Despite this, there is as yet little known about the isoform specific distribution in neurons. RESULTS: With help of superresolving stimulated emission depletion microscopy the spatial distribution of Na+,K+-ATPase in dendritic spines of cultured striatum neurons have been dissected. The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (α3 isoform) in the postsynaptic region of the spine. CONCLUSIONS: A compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins. Superresolution microscopy has thus opened up a new perspective to elucidate the nature of the physiological function, regulation and signaling role of Na+,K+-ATPase from its topological distribution in dendritic spines.

Localization of Na+-K+ ATPases in quasi-native cell membranes.

Na(+)-K(+) ATPases have been observed and located by in situ AFM and single molecule recognition technique, topography and recognition imaging (TREC) that is a unique technique to specifically identify single protein in complex during AFM imaging. Na(+)-K(+) ATPases were well distributed in the inner leaflet of cell membranes with about 10% aggregations in total recognized proteins. The height of Na(+)-K(+) ATPases measured by AFM is in the range of 12-14 nm, which is very consistent with the cryoelectron microscopy result. The unbinding force between Na(+)-K(+) ATPases in the membrane and anti-ATPases on the AFM tip is about 80 pN with the apparent loading rate at 40 nN/s. Our results show the first visualization of an essential membrane protein, Na(+)-K(+) ATPase, in quasi-native cell membranes and may be significant to reveal the interactions between Na(+)-K(+) ATPases and other membrane proteins at the molecular level.

[Energetic applications: Na+/K+-ATPase and neuromuscular transmission]. / Applications énergétiques: Na/K-ATPase et transmission neuromusculaire.

Na/K-ATPase electrogenic activity and its indispensable role in maintaining gradients suggest that the modifications in isoform distribution and the functioning of the sodium pump have a major influence on both the neuronal functions, including excitability, and motor efficiency. This article proposes to clarify the involvement of Na/K-ATPase in the transmission of nerve influx within the peripheral nerve and then in the genesis, the maintenance, and the physiology of muscle contraction by comparing the data found in the literature with our work on neuron and muscle characterization of Na/K-ATPase activity and isoforms.

Osmotic stress and viscous retardation of the Na,K-ATPase ion pump.

The transport function of the Na pump (Na,K-ATPase) in cellular ion homeostasis involves both nucleotide binding reactions in the cytoplasm and alternating aqueous exposure of inward- and outward-facing ion binding sites. An osmotically active, nonpenetrating polymer (poly(ethyleneglycol); PEG) and a modifier of the aqueous viscosity (glycerol) were used to probe the overall and partial enzymatic reactions of membranous Na,K-ATPase from shark salt glands. Both inhibit the steady-state Na,K-ATPase as well as Na-ATPase activity, whereas the K(+)-dependent phosphatase activity is little affected by up to 50% of either. Both Na,K-ATPase and Na-ATPase activities are inversely proportional to the viscosity of glycerol solutions in which the membranes are suspended, in accordance with Kramers' theory for strong coupling of fluctuations at the active site to solvent mobility in the aqueous environment. PEG decreases the affinity for Tl(+) (a congener for K(+)), whereas glycerol increases that for the nucleotides ATP and ADP in the presence of NaCl but has little effect on the affinity for Tl(+). From the dependence on osmotic stress induced by PEG, the aqueous activation volume for the Na,K-ATPase reaction is estimated to be approximately 5-6 nm(3) (i.e., approximately 180 water molecules), approximately half this for Na-ATPase, and essentially zero for p-nitrophenol phosphatase. The change in aqueous hydrated volume associated with the binding of Tl(+) is in the region of 9 nm(3). Analysis of 15 crystal structures of the homologous Ca-ATPase reveals an increase in PEG-inaccessible water space of approximately 22 nm(3) between the E(1)-nucleotide bound forms and the E(2)-thapsigargin forms, showing that the experimental activation volumes for Na,K-ATPase are of a magnitude comparable to the overall change in hydration between the major E(1) and E(2) conformations of the Ca-ATPase.

Conduction of Na+ and K+ through the NaK channel: molecular and Brownian dynamics studies.

Conduction of ions through the NaK channel, with M0 helix removed, was studied using both Brownian dynamics and molecular dynamics. Brownian dynamics simulations predict that the truncated NaK has approximately a third of the conductance of the related KcsA K+ channel, is outwardly rectifying, and has a Michaelis-Menten current-concentration relationship. Current magnitude increases when the glutamine residue located near the intracellular gate is replaced with a glutamate residue. The channel is blocked by extracellular Ca2+. Molecular dynamics simulations show that, under the influence of a strong applied potential, both Na+ and K+ move across the selectivity filter, although conduction rates for Na+ ions are somewhat lower. The mechanism of conduction of Na+ differs significantly from that of K+ in that Na+ is preferentially coordinated by single planes of pore-lining carbonyl oxygens, instead of two planes as in the usual K+ binding sites. The water-containing filter pocket resulting from a single change in the selectivity filter sequence (compared to potassium channels) disrupts several of the planes of carbonyl oxygens, and thus reduces the filter's ability to discriminate against sodium.

Three-dimensional structure of the KdpFABC complex of Escherichia coli by electron tomography of two-dimensional crystals.

The KdpFABC complex (Kdp) functions as a K+ pump in Escherichia coli and is a member of the family of P-type ATPases. Unlike other family members, Kdp has a unique oligomeric composition and is notable for segregating K+ transport and ATP hydrolysis onto separate subunits (KdpA and KdpB, respectively). We have produced two-dimensional crystals of the KdpFABC complex within reconstituted lipid bilayers and determined its three-dimensional structure from negatively stained samples using a combination of electron tomography and real-space averaging. The resulting map is at a resolution of 2.4 nm and reveals a dimer of Kdp molecules as the asymmetric unit; however, only the cytoplasmic domains are visible due to the lack of stain penetration within the lipid bilayer. The sizes of these cytoplasmic domains are consistent with Kdp and, using a pseudo-atomic model, we have described the subunit interactions that stabilize the Kdp dimer within the larger crystallographic array. These results illustrate the utility of electron tomography in structure determination of ordered assemblies, especially when disorder is severe enough to hamper conventional crystallographic analysis.

Localization and irregular distribution of Na,K-ATPase in myelin sheath from rat sciatic nerve.

Sodium, potassium adenosine triphosphatase (Na,K-ATPase) is a membrane-bound enzyme that maintains the Na(+) and K(+) gradients used in the nervous system for generation and transmission of bioelectricity. Recently, its activity has also been demonstrated during nerve regeneration. The present study was undertaken to investigate the ultrastructural localization and distribution of Na,K-ATPase in peripheral nerve fibers. Small blocks of the sciatic nerves of male Wistar rats weighing 250-300g were excised, divided into two groups, and incubated with and without substrate, the para-nitrophenyl phosphate (pNPP). The material was processed for transmission electron microscopy, and the ultra-thin sections were examined in a Philips CM 100 electron microscope. The deposits of reaction product were localized mainly on the axolemma, on axoplasmic profiles, and irregularly dispersed on the myelin sheath, but not in the unmyelinated axons. In the axonal membrane, the precipitates were regularly distributed on the cytoplasmic side. These results together with published data warrant further studies for the diagnosis and treatment of neuropathies with compromised Na,K-ATPase activity.

Determination of the sidedness of the carboxy-terminus of the Na+/K(+)ATPase alpha-subunit using lactoperoxidase iodination.

The orientation of the carboxy-terminal pair of tyrosines of the Na+/K(+)-ATPase alpha-subunit with respect to the plane of the plasma membrane was determined. The approach was based on lactoperoxidase-catalysed radioiodination of the tyrosine residues accessible on the surface of the enzyme molecule in intact cells of a pig kidney embryonic cell line and those accessible in a broken plasma membrane fraction and in isolated membrane-bound Na+/K(+)-ATPase. The labeled alpha-subunit was isolated by SDS gel electrophoresis followed by electroblotting. Then the COOH-terminal amino acids were hydrolyzed by carboxypeptidases B and Y. Radioactivity and quantitative analysis of the protein and released amino acids showed that the COOH-terminal tyrosine residues of the alpha-subunit were only accessible to modification only when lactoperoxidase had access to the inner side of the plasma membrane. Therefore, the COOH-terminus of the Na+/K(+)-ATPase alpha-subunit is located on the cytoplasmic surface of the pump molecule and its polypeptide chain must have an even number of transmembrane segments.

Three-dimensional structure of renal Na,K-ATPase from cryo-electron microscopy of two-dimensional crystals.

The structure of Na, K-ATPase was determined by electron crystallography at 9.5 A from multiple small 2-D crystals induced in purified membranes isolated from the outer medulla of pig kidney. The density map shows a protomer stabilized in the E(2) conformation which extends approximately 65 A x 75 A x 150 A in the asymmetric unit of the P2 type unit cell. The alpha, beta, and gamma subunits were demonstrated in the membrane crystals with Western blotting and related to distinct domains in the density map. The alpha subunit corresponds to most of the density in the transmembrane region as well as the large hydrophilic headpiece on the cytoplasmic side of the membrane. The headpiece is divided into three separated domains, which are similar in overall shape to the domains of the calcium pump of the sarcoplasmic reticulum. One of these domains gives rise to a characteristic elongated projection onto the membrane plane while the putative nucleotide binding and phosphorylation domains form comparatively compact densities in the rest of the cytoplasmic part of the structure. Density on the extracellular face corresponds to the protein part of the beta subunit and is located as an extension of the transmembrane region perpendicular to the membrane plane. The structure of the lipid bilayer spanning part suggests the positions for the transmembrane helix from the beta subunit as well as the small gamma subunit present in this Na,K-ATPase. Two groups of ten helices from the catalytic alpha subunit corresponds to the remaining density in the transmembrane region. The present results demonstrate distinct similarities between the structure of the alpha subunit of Na,K-ATPase as determined here by cryo-electron microscopy and the reported X-ray structure of Ca-ATPase. However, conformational changes between the E(1) and E(2) forms are suggested by different relative positions of cytoplasmatic domains.

Branchial chamber tissues in two caridean shrimps: the epibenthic Palaemon adspersus and the deep-sea hydrothermal Rimicaris exoculata.

The structure of the epithelia of the branchial chamber organs (gills, branchiostegites, epipodites) and the localization of the Na(+),K(+)-ATPase were investigated in two caridean shrimps, the epibenthic Palaemon adspersus and the deep-sea hydrothermal Rimicaris exoculata. The general organization of the phyllobranchiate gills, branchiostegites and epipodites is similar in P. adspersus and in R. exoculata. The gill filaments are formed by a single axial epithelium made of H-shaped cells with thin lateral expansions and a basal lamina limiting hemolymph lacunae. In P. adspersus, numerous ionocytes are present in the epipodites and in the inner-side of the branchiostegites; immunofluorescence reveals their high content in Na(+),K(+)-ATPase. In R. exoculata, typical ionocytes displaying a strong Na(+),K(+)-ATPase specific fluorescence are observed in the epipodites only. While the epipodites and the branchiostegites appear as the main site of osmoregulation in P. adspersus, only the epipodites might be involved in ion exchanges in R. exoculata. In both species, the gill filaments are mainly devoted to respiration.

Crystal structure of a light-driven sodium pump.

Recently, the first known light-driven sodium pumps, from the microbial rhodopsin family, were discovered. We have solved the structure of one of them, Krokinobacter eikastus rhodopsin 2 (KR2), in the monomeric blue state and in two pentameric red states, at resolutions of 1.45 Å and 2.2 and 2.8 Å, respectively. The structures reveal the ion-translocation pathway and show that the sodium ion is bound outside the protein at the oligomerization interface, that the ion-release cavity is capped by a unique N-terminal α-helix and that the ion-uptake cavity is unexpectedly large and open to the surface. Obstruction of the cavity with the mutation G263F imparts KR2 with the ability to pump potassium. These results pave the way for the understanding and rational design of cation pumps with new specific properties valuable for optogenetics.

Ultrastructure of membrane-bound Na, K-ATPase after extensive tryptic digestion.

Membrane-bound Na, K-ATPase was digested with trypsin in the presence of Rb+ to form the stable 19-kDa and smaller fragments of the alpha-chain known to preserve occlusion of Rb+ (K+) or Na+. The trypsinized membranes obtained from pig kidney and shark rectal gland were analyzed by electron microscopy. Tryptic digestion preserved general membrane structure but removed both the surface particles observed by negative staining and the protruding cytoplasmic portion of the alpha-subunit identified in thin sections. However, intramembrane particles defined by freeze-fracture were preserved after trypsinization suggesting that the remaining membrane spanning protein fragments retain the native structure within the lipid bilayer after proteolysis.

A pH induced two-dimensional crystal of membrane-bound Na+,K(+)-ATPase of dog kidney.

Two-dimensional crystals of membrane-bound Na+,K(+)-ATPase were formed in acidic media and their qualities were investigated by electron cryo-microscopy as well as by conventional electron microscopy. At pH 4.8 in sodium citrate buffer, the best crystallization condition, more than 80% of membranes formed crystals. The high ratio allowed high-resolution images to be taken by electron cryo-microscopy. Image processing revealed that they had unique lattice constants (a = 108.7 A, b = 66.2 A, gamma = 104.2 degrees) and had few defects in the crystalline arrays. The reconstituted Fourier map of the ice-embedded crystal showed that there are two high contrast parts in one unit cell.

Molecular imaging of Na+,K(+)-ATPase in purified kidney membranes.

Ion channels and pumps in cell membranes consist of multiple transmembrane segments that are thought to be critical for transport of ions. Channel structures constituted by these transmembrane segments are characteristic of ion channels, whereas such structures have not been identified in ion pumps until now. By applying atomic force microscopy on Na+,K(+)-ATPase molecules in canine kidney membranes under tapping mode, we identified a hollow in the protein with a characteristic internal diameter of 6-20 A and an external diameter of 20-55 A depending upon treatment conditions. This hollow may be interpreted as a channel-like conformation of Na+,K(+)-ATPase. In the regions where the proteins were absent, lipid head structures with 2 A width and 6 A length were imaged in an orthorhombic lattice.

Ultrastructural localization of Na+, K(+)-ATPase in the exorbital lacrimal gland of rat.

Ultrastructural localization of Na+, K(+)-ATPase in the exorbital lacrimal gland of rat was investigated quantitatively by protein A-gold technique. Na+, K(+)-ATPase was purified from the rat kidney, and anti-holo Na+, K(+)-ATPase antibody was obtained from the rabbit by injecting the purified enzyme. A specific antibody against the alpha-subunit of Na+, K(+)-ATPase was affinity purified. Immunoblot analysis revealed that the antibody bound specifically to the alpha-subunit of Na+, K(+)-ATPase of the lacrimal gland. Rats were fixed by perfusion with 4% paraformaldehyde containing 1% glutaraldehyde, and the lacrimal glands were embedded in LR White resin. Ultrathin sections were incubated with affinity purified antibody against the alpha-subunit of Na+, K(+)-ATPase, and then with protein A-gold complex. The sections were observed under an electron microscope. Light microscopy with silver enhancement procedure revealed that Na+, K(+)-ATPase was located mainly on the basal region of the cells of intralobular and interlobular ducts. Quantitative immunoelectron microscopic analysis showed that gold particles were found on the basolateral surfaces of the interlobular and intralobular ducts cells and on the basolateral surface of the acinar cells, whereas no significant binding was observed on any part of the apical surfaces of these cells. Labeling density of gold particles was highest on the basolateral surface of the interlobular duct cells, secondarily highest on the basolateral surface of the intralobular duct cells, and lowest on the basolateral surface of the acinar cells. The distribution pattern of Na+, K(+)-ATPase in the acinar cells and the duct cells suggest that this enzyme may play an important role in primary secretion and in determining the composition of electrolytes in the final secretion, respectively.

The oligomeric nature of Na/K-transport ATPase.

Since the discovery of Na/K-ATPase, evidence has accumulated to suggest that 1 mol of ATP hydrolysis occurs via the Na(+)-occluded ADP-sensitive phosphoenzyme, the K(+)-sensitive phosphoenzyme and the K(+)-occluded enzyme accompanying active transport of 3Na(+) and 2K(+) according the Post-Albers scheme. However, some controversial issues have arisen concerning whether the functional unit of the enzyme is an alpha beta-protomer or a much higher oligomer, which would be related to the mechanism of transport, either sequential or simultaneous. Detailed studies of oligomer interaction and the reactivity of the enzyme and a comparison of the extent of phosphorylation with ligand-binding capacities in the presence or absence of ATP hydrolysis and others strongly suggest that the functional unit of the enzyme in the membrane is a tetraprotomer, (alpha beta)(4). They also suggest that each reaction intermediate of the Post-Albers scheme, respectively, reflects half of the site property of the intermediate and that another half binds ATP. These data may be useful not only to answer the long-standing question of whether the mechanism functions in the presence of both Na(+) and K(+) but also contribute to a better understanding of the mechanism of P-type pump ATPase in general.

Renal Na,K-ATPase structure from cryo-electron microscopy of two-dimensional crystals.

The molecular structure of Na,K-ATPase was determined by electron crystallography from two-dimensional crystals induced in purified membranes isolated from the outer medulla of pig kidney. The P2 type unit cell contains two protomers in the E(2) conformation, each of them with a size of 65 x 75 x 150 A(3). The alpha, beta, and gamma subunits in the membrane crystals were demonstrated in the crystals with Western blotting and related to distinct domains in the density map. The alpha subunit corresponds to most of the density in the transmembrane region as well as to the large hydrophilic headpiece on the cytoplasmic side of the membrane. The headpiece is divided into three separated domains. One of these gives rise to an elongated projection onto the membrane plane, while the putative nucleotide binding and phosphorylation domains form compact densities in the rest of the cytoplasmic part of the structure. Density on the extracellular face corresponds to the protein part of the beta subunit. Ten helices from the catalytic a subunit correspond to two groups of distinct densities in the transmembrane region. The structure of the lipid bilayer spanning part also suggests positions for the transmembrane helices from the beta and gamma subunits. The overall structure of the alpha subunit of Na,K-ATPase as determined here by cryo-electron microscopy is similar to the X-ray structure of Ca-ATPase. However, conformational changes between the E(1) and E(2) forms are suggested by different relative positions of cytoplasmic domains.