K+ congeners that do not compromise Na+ activation of the Na+,K+-ATPase: hydration of the ion binding cavity likely controls ion selectivity.

The Na(+),K(+)-ATPase is essential for ionic homeostasis in animal cells. The dephosphoenzyme contains Na(+) selective inward facing sites, whereas the phosphoenzyme contains K(+) selective outward facing sites. Under normal physiological conditions, K(+) inhibits cytoplasmic Na(+) activation of the enzyme.

Identification of electric-field-dependent steps in the Na(+),K(+)-pump cycle.

The charge-transporting activity of the Na(+),K(+)-ATPase depends on its surrounding electric field. To isolate which steps of the enzyme's reaction cycle involve charge movement, we have investigated the response of the voltage-sensitive fluorescent probe RH421 to interaction of the protein with BTEA (benzyltriethylammonium), which binds from the extracellular medium to the Na(+),K(+)-ATPase's transport sites in competition with Na(+) and K(+), but is not occluded within the protein. We find that only the occludable ions Na(+), K(+), Rb(+), and Cs(+) cause a drop in RH421 fluorescence.

Inhibition of K+ transport through Na+, K+-ATPase by capsazepine: role of membrane span 10 of the α-subunit in the modulation of ion gating.

Capsazepine (CPZ) inhibits Na+,K+-ATPase-mediated K+-dependent ATP hydrolysis with no effect on Na+-ATPase activity. In this study we have investigated the functional effects of CPZ on Na+,K+-ATPase in intact cells. We have also used well established biochemical and biophysical techniques to understand how CPZ modifies the catalytic subunit of Na+,K+-ATPase.

Susceptibility of β1 Na+-K+ pump subunit to glutathionylation and oxidative inhibition depends on conformational state of pump.

Glutathionylation of cysteine 46 of the β1 subunit of the Na(+)-K(+) pump causes pump inhibition. However, the crystal structure, known in a state analogous to an E2·2K(+)·P(i) configuration, indicates that the side chain of cysteine 46 is exposed to the lipid bulk phase of the membrane and not expected to be accessible to the cytosolic glutathione. We have examined whether glutathionylation depends on the conformational changes in the Na(+)-K(+) pump cycle as described by the Albers-Post scheme.

Stabilization of the α2 isoform of Na,K-ATPase by mutations in a phospholipid binding pocket.

The α2 isoform of Na,K-ATPase plays a crucial role in Ca(2+) handling, muscle contraction, and inotropic effects of cardiac glycosides. Thus, structural, functional, and pharmacological comparisons of α1, α2, and α3 are of great interest. In Pichia pastoris membranes expressing human α1β1, α2β1, and α3β1 isoforms, or using the purified isoform proteins, α2 is most easily inactivated by heating and detergent (α2 ≫ α3 > α1).

Oleic and linoleic acids are active principles in Nigella sativa and stabilize an E(2)P conformation of the Na,K-ATPase. Fatty acids differentially regulate cardiac glycoside interaction with the pump.

Nigella sativa seed oil was found to contain a modulator of Na,K-ATPase. Separation analyses combined with (1)H NMR and GCMS identified the inhibitory fraction as a mixture of oleic and linoleic acids. These two fatty acids are specifically concentrated in several medicinal plant oils, and have particularly been implicated in decreasing high blood pressure.

Metal fluoride complexes of Na,K-ATPase: characterization of fluoride-stabilized phosphoenzyme analogues and their interaction with cardiotonic steroids.

The Na,K-ATPase belongs to the P-type ATPase family of primary active cation pumps. Metal fluorides like magnesium-, beryllium-, and aluminum fluoride act as phosphate analogues and inhibit P-type ATPases by interacting with the phosphorylation site, stabilizing conformations that are analogous to specific phosphoenzyme intermediates. Cardiotonic steroids like ouabain used in the treatment of congestive heart failure and arrhythmias specifically inhibit the Na,K-ATPase, and the detailed structure of the highly conserved binding site has recently been described by the crystal structure of the shark Na,K-ATPase in a state analogous to E2·2K(+)·P(i) with ouabain bound with apparently low affinity (1).

Curcumin is a lipid dependent inhibitor of the Na,K-ATPase that likely interacts at the protein-lipid interface.

Curcumin is an important nutraceutical widely used in disease treatment and prevention. We have previously suggested that curcumin interferes with K(+) binding to pig kidney Na,K-ATPase by interaction with its extracellular domains. The aim of this study was to further characterize the site of curcumin interaction with the ATPase.

FXYD-11 associates with Na+-K+-ATPase in the gill of Atlantic salmon: regulation and localization in relation to changed ion-regulatory status.

The Na(+)-K(+)-ATPase is the primary electrogenic component driving transepithelial ion transport in the teleost gill; thus regulation of its level of activity is of critical importance for osmotic homeostasis. In the present study, we examined the dynamics of the gill-specific FXYD-11 protein, a putative regulatory subunit of the pump, in Atlantic salmon during seawater (SW) acclimation, smoltification, and treatment with cortisol, growth hormone, and prolactin. Dual-labeling immunohistochemistry showed that branchial FXYD-11 is localized in Na(+)-K(+)-ATPase immunoreactive cells, and coimmunoprecipitation experiments confirmed a direct association between FXYD-11 and the Na(+)-K(+)-ATPase α-subunit.

Interaction between cardiotonic steroids and Na,K-ATPase. Effects of pH and ouabain-induced changes in enzyme conformation.

The Na,K-ATPase belongs to the P-type ATPase family of primary active cation pumps. It maintains the transmembrane gradients of Na(+) and K(+) across the cell membrane essential for cell homeostasis. The Na,K-ATPase is specifically inhibited by cardiotonic steroids like ouabain, which bind to the extracellular side of the enzyme and is of significant therapeutic value in the treatment of congestive heart failure.

Capsaicin stimulates uncoupled ATP hydrolysis by the sarcoplasmic reticulum calcium pump.

In muscle cells the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) couples the free energy of ATP hydrolysis to pump Ca(2+) ions from the cytoplasm to the SR lumen. In addition, SERCA plays a key role in non-shivering thermogenesis through uncoupled reactions, where ATP hydrolysis takes place without active Ca(2+) translocation. Capsaicin (CPS) is a naturally occurring vanilloid, the consumption of which is linked with increased metabolic rate and core body temperature.

Capsazepine, a synthetic vanilloid that converts the Na,K-ATPase to Na-ATPase.

Capsazepine (CPZ), a synthetic capsaicin analogue, inhibits ATP hydrolysis by Na,K-ATPase in the presence but not in the absence of K(+). Studies with purified membranes revealed that CPZ reduced Na(+)-dependent phosphorylation by interference with Na(+) binding from the intracellular side of the membrane. Kinetic analyses showed that CPZ stabilized an enzyme species that constitutively occluded K(+).

Modulation of FXYD interaction with Na,K-ATPase by anionic phospholipids and protein kinase phosphorylation.

FXYD10 is a 74 amino acid small protein which regulates the activity of shark Na,K-ATPase. The lipid dependence of this regulatory interaction of FXYD10 with shark Na,K-ATPase was investigated using reconstitution into DOPC/cholesterol liposomes with or without the replacement of 20 mol % DOPC with anionic phospholipids. Specifically, the effects of the cytoplasmic domain of FXYD10, which contains the phosphorylation sites for protein kinases, on the kinetics of the Na,K-ATPase reaction were investigated by a comparison of the reconstituted native enzyme and the enzyme where 23 C-terminal amino acids of FXYD10 had been cleaved by mild, controlled trypsin treatment.

Stabilization of trypsin by association to plasma membranes: implications for tryptic cleavage of membrane-bound Na,K-ATPase.

Tryptic cleavage has been a potential method for studying the structure and mechanism of many membrane transport proteins. Here, we report tight association of trypsin to pig kidney plasma membranes enriched in Na,K-ATPase. Trypsin also associated with protein-free vesicles prepared from plasma membrane lipids.

Functional significance of the shark Na,K-ATPase N-terminal domain. Is the structurally variable N-Terminus involved in tissue-specific regulation by FXYD proteins?

The proteolytic profile after mild controlled trypsin cleavage of shark rectal gland Na,K-ATPase was characterized and compared to that of pig kidney Na,K-ATPase, and conditions for achieving N-terminal cleavage of the alpha-subunit at the T(2) trypsin cleavage site were established. Using such conditions, the shark enzyme N-terminus was much more susceptible to proteolysis than the pig enzyme. Nevertheless, the maximum hydrolytic activity was almost unaffected for the shark enzyme, whereas it was significantly decreased for the pig kidney enzyme.

Interaction of FXYD10 (PLMS) with Na,K-ATPase from shark rectal glands. Close proximity of Cys74 of FXYD10 to Cys254 in the a domain of the alpha-subunit revealed by intermolecular thiol cross-linking.

FXYD domain-containing proteins are tissue-specific regulators of the Na,K-ATPase that have been shown to have significant physiological implications. Information about the sites of interaction between some FXYD proteins and subunits of the Na,K-ATPase is beginning to emerge. We previously identified an FXYD protein in plasma membranes from shark rectal gland cells and demonstrated that this protein (FXYD10) modulates shark Na,K-ATPase activity.

Curcumin modulation of Na,K-ATPase: phosphoenzyme accumulation, decreased K+ occlusion, and inhibition of hydrolytic activity.

1 Curcumin, the major constitute of tumeric, is an important nutraceutical that has been shown to be useful in the treatment of many diseases. As an inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase, curcumin was shown to correct cystic fibrosis (CF) defects in some model systems, whereas others have reported no or little effects on CF after curcumin treatment, suggesting that curcumin effect is not due to simple inhibition of the Ca(2+)-ATPase. 2 We tested the hypothesis that curcumin may modulate other members of the P(2)-type ATPase superfamily by studying the effects of curcumin on the activity and kinetic properties of the Na,K-ATPase.

Functional modulation of the sodium pump: the regulatory proteins "Fixit".

Proteins of the FXYD family act as tissue-specific regulators of the Na-K-ATPase. They are small hydrophobic type I proteins with a single-transmembrane span containing an extracellular invariant FXYD sequence. FXYD proteins are not an integral part of the Na-K-ATPase but function to modulate its catalytic properties by molecular interactions with specific Na-K-ATPase domains.

Direct activation of gastric H,K-ATPase by N-terminal protein kinase C phosphorylation. Comparison of the acute regulation mechanisms of H,K-ATPase and Na,K-ATPase.

In this study we compared the protein kinase dependent regulation of gastric H,K-ATPase and Na,K-ATPase. The protein kinase A/protein kinase C (PKA/PKC) phosphorylation profile of H,K-ATPase was very similar to the one found in the Na,K-ATPase. PKC phosphorylation was taking place in the N-terminal part of the alpha-subunit with a stoichiometry of approximately 0.

Protein kinase C phosphorylation of purified Na,K-ATPase: C-terminal phosphorylation sites at the alpha- and gamma-subunits close to the inner face of the plasma membrane.

The alpha-subunit of the Na,K-ATPase is phosphorylated at specific sites by protein kinases A and C. Phosphorylation by protein kinase C (PKC) is restricted to the N terminus and takes place to a low stoichiometry, except in rat. Here we show that the alpha-subunit of shark Na,K-ATPase can be phosphorylated by PKC at C-terminal sites to stoichiometric levels in the presence of detergents.