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Na+,K+‐ATPase (NKA) plays a pivotal role in establishing electrochemical gradients for Na+ and K+ across the cell membrane by alternating between the E1 (showing high affinity for Na+ and low affinity for K+) and E2 (low affinity to Na+ and high affinity to K+) forms. Presented here are two crystal structures of NKA in E1·Mg2+ and E1·3Na+ states at 2.9 and 2.8 Å resolution, respectively. These two E1 structures fill a gap in our description of the NKA reaction cycle based on the atomic structures. We describe how NKA converts the K+‐bound E2·2K+ form to an E1 (E1·Mg2+) form, which allows high‐affinity Na+ binding, eventually closing the cytoplasmic gate (in E1 ~ P·ADP·3Na+) after binding three Na+, while keeping the extracellular ion pathway sealed. We now understand previously unknown functional roles for several parts of NKA and that NKA uses even the lipid bilayer for gating the ion pathway. [ABSTRACT FROM AUTHOR]

Na+,K+‐ATPase (NKA) is one of the most important members of the P‐type ion‐translocating ATPases and plays a pivotal role in establishing electrochemical gradients for Na+ and K+ across the cell membrane. Presented here is a 3.3 Å resolution structure of NKA in the E2·2K+ state solved by cryo‐electron microscopy. It is a stable state with two occluded K+ after transferring three Na+ into the extracellular medium and releasing inorganic phosphate bound to the cytoplasmic P domain. We describe how the extracellular ion pathway wide open in the E2P state becomes closed and locked in E2·2K+, linked to events at the phosphorylation site more than 50 Å away. We also show, although at low resolution, how ATP binding to NKA in E2·2K+ relaxes the gating machinery and thereby accelerates the transition into the next step, that is, the release of K+ into the cytoplasm, more than 100 times. [ABSTRACT FROM AUTHOR]

[Na.sup.+], [K.sup.+] -ATPase pumps three [Na.sup.+] ions out of cells in exchange for two [K.sup.+] taken up from the extracellular medium per ATP molecule hydrolysed, thereby establishing [Na.sup.+] and [K.sup.+] gradients across the membrane in all animal cells. These ion gradients are used in many fundamental processes, notably excitation of nerve cells. Here we describe 2.8 Å- resolution crystal structures of this ATPase from pig kidney with bound [Na.sup.+], ADP and aluminium fluoride, a stable phosphate analogue, with and without oligomycin that promotes [Na.sup.+] occlusion. These crystal structures represent a transition state preceding the phosphorylated intermediate (E1P) in which three [Na.sup.+] ions are occluded. Details of the [Na.sup.+] -binding sites show how this ATPase functions as a [Na.sup.+]-specific pump, rejecting [K.sup.+] and Ca2+, even though its affinity for [Na.sup.+] is low (millimolar dissociation constant). A mechanism for sequential, cooperative [Na.sup.+] binding can now be formulated in atomic detail., [Na.sup.+],[K.sup.+]-ATPase is expressed in all animal cells and is one of the most important members of the P-type ion-translocating ATPases that include [Ca.sup.2+]-ATPase of fast-twitch muscle sarcoplasmic reticulum (SERCA1a) and [...]

Sodium-potassium ATPase is an ATP-powered ion pump that establishes concentration gradients for [Na.sub.+] and [K.sub.+] ions across the plasma membrane in all animal cells by pumping [Na.sub.+] from the cytoplasm [...]

The effects of phospholipids on the functional regulation of shark Na, K-ATPase by FXYD10 were examined. The findings show that phosphorylation involves only modest structural rearrangements between the cytoplasmic domain of FXYD10 and the Na, K-ATPase A-domain.

In this investigation we assess the potential of Raman spectroscopy as a tool for probing conformational changes in membrane-spanning proteins--in this case, the sodium potassium adenosine triphosphatase ([Na.sup.+],[K.sup.+]-ATPase). Spectral analysis […]

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. It was suggested that phospholemman-like protein from shark (PLMS) interacts functionally with the N-terminus of the shark Na,K-ATPase to control the E1-E2 conformational transition of the enzyme and that such interactions may be controlled by regulatory protein kinase phosphorylation of the N-terminus.

Cryoelectron microscopy (cryo-EM) was applied to Na+,K+-ATPase (NKA) to determine the structures of two E2P states, one (E2PATP) formed by ATP and Mg2+ in the forward reaction, and the other (E2PPi) formed by inorganic phosphate (Pi) andMg2+ in the backward reaction, with and without ouabain or istaroxime, representatives of classical and new-generation cardiotonic steroids (CTSs). These two E2P states exhibit different biochemical properties. In particular, K+-sensitive acceleration of the dephosphorylation reaction is not observed with E2PPi, attributed to the presence of a Mg2+ ion in the transmembrane cation binding sites. The cryo-EM structures of NKA demonstrate that the two E2P structures are nearly identical but Mg2+ in the transmembrane binding cavity is identified only in E2PPi, corroborating the idea that it should be denoted as E2PPi.Mg2+. We can now explain why the absence of transmembrane Mg2+ in E2PATP confers the K+ sensitivity in dephosphorylation. In addition, we show that ATP bridges the actuator (A) and nucleotide binding (N) domains, stabilizing the E2PATP state; CTS binding causes hardly any changes in the structure of NKA, both in E2PATP and E2PPi.Mg2+, indicating that the binding mechanism is conformational selection; and istaroxime binds to NKA, extending its aminoalkyloxime group deep into the cation binding site. This orientation is upside down compared to that of classical CTSs with respect to the steroid ring. Notably, mobile parts of NKA are resolved substantially better in the electron microscopy (EM) maps than in previous X-ray structures, including sugars sticking out from the ß-subunit and many phospholipid molecules. [ABSTRACT FROM AUTHOR]

The seven mammalian FXYD proteins associate closely with α/β heterodimers of Na+/K+-ATPase. Most of them protect the β1 subunit against glutathionylation, an oxidative modification that destabilizes the heterodimer and inhibits Na+/K+-ATPase activity. A specific cysteine (Cys) residue of FXYD proteins is critical for such protection. One of the FXYD proteins, FXYD3, confers treatment resistance when overexpressed in cancer cells. We developed two FXYD3 peptide derivatives. FXYD3-pep CKCK retained the Cys residue that can undergo glutathionylation and that is critical for protecting the β1 subunit against glutathionylation. FXYD3-pep SKSK had all Cys residues mutated to Serine (Ser). The chemotherapeutic doxorubicin induces oxidative stress, and suppression of FXYD3 with siRNA in pancreatic- and breast cancer cells that strongly express FXYD3 increased doxorubicin-induced cytotoxicity. Exposing cells to FXYD3-pep SKSK decreased co-immunoprecipitation of FXYD3 with the α1 Na+/K+-ATPase subunit. FXYD3-pep SKSK reproduced the increase in doxorubicin-induced cytotoxicity seen after FXYD3 siRNA transfection in pancreatic- and breast cancer cells that overexpressed FXYD3, while FXYD3-pep CKCK boosted the native protein's protection against doxorubicin. Neither peptide affected doxorubicin's cytotoxicity on cells with no or low FXYD3 expression. Fluorescently labeled FXYD3-pep SKSK was detected in a perinuclear distribution in the cells overexpressing FXYD3, and plasmalemmal Na+/K+-ATPase turnover could not be implicated in the increased sensitivity to doxorubicin that FXYD3-pep SKSK caused. FXYD peptide derivatives allow rapid elimination or amplification of native FXYD protein function. Here, their effects implicate the Cys residue that is critical for countering β1 subunit glutathionylation in the augmentation of cytotoxicity with siRNA-induced downregulation of FXYD3. [ABSTRACT FROM AUTHOR]

Research has been conducted on the unphosphorylated Na (super)+, K (super)+ -ATPase. The kinetics of the conformational change of this ATPase has been investigated via the use of the stopped-flow technique and the results suggest that Na (super)+ ions play a regulatory role in enhancement of the conformational transition rate.

Research has been conducted on the phospholipid acyl chain length. The effects of this chain length, the chain saturation degree and cholecterol on the Na,K-ATPase liposome reconstitution have been investigated.

Research was conducted to examine the characteristics of Na,K-ATPase phosphoenzymes formed either from ATP in the presence of Mg2+ and Na+ or from P(sub i) in the absence of alkali cations. Spectrofluorometry and biochemical methods using the styril dye RH421 were used for the investigation. Results indicate that the increase of RH421 fluorescence induced by inorganic phosphate in the absence of alkali cations is associated with the formation of the covalent acyl-phosphate bond.

The structure of the styryl-based RH dyes regulates the fluorescence response of Na,K-ATPase towards phosphorylation. The net charge of the dye molecule is a critical factor for determining the sensitivity and the spectral properties of the dyes response to phophorylation. Computer modeling of dyes with the same chromophore structure reveals that the absorption maximum of the zwitterionic dyes is blue-shifted and the quantum yield of these dyes is higher than that for the positively charged dyes. The electrostatic interactions between the hydrophilic headgroup of the dye and the enzyme change the net charge of the dye molecule.

The sodium pump (Na+, K+-ATPase, NKA) is vital for animal cells, as it actively maintains Na+ and K+ electrochemical gradients across the cell membrane. It is a target of cardiotonic steroids (CTSs) such as ouabain and digoxin. As CTSs are almost unique strong inhibitors specific to NKA, a wide range of derivatives has been developed for potential therapeutic use. Several crystal structures have been published for NKA-CTS complexes, but they fail to explain the largely different inhibitory properties of the various CTSs. For instance, although CTSs are thought to inhibit ATPase activity by binding to NKA in the E2P state, we do not know if large conformational changes accompany binding, as no crystal structure is available for the E2P state free of CTS. Here, we describe crystal structures of the BeF3- complex of NKA representing the E2P ground state and then eight crystal structures of seven CTSs, including rostafuroxin and istaroxime, two new members under clinical trials, in complex with NKA in the E2P state. The conformations of NKA are virtually identical in all complexes with and without CTSs, showing that CTSs bind to a preformed cavity in NKA. By comparing the inhibitory potency of the CTSs measured under four different conditions, we elucidate how different structural features of the CTSs result in different inhibitory properties. The crystal structures also explain K+-antagonism and suggest a route to isoform specific CTSs. [ABSTRACT FROM AUTHOR]

The voltage-sensitive fluorescent probe RH421 was used to study the interaction of ATP with the phosphoenzyme of Na(+),K(+)-ATPase from pig kidney, rabbit kidney, and shark rectal gland. The results provide insight into the possible physiological role of allosteric binding of ATP to the phosphoenzyme towards the stabilization of the E2P state and avoid the enzyme running backward which would cause dissipation of the Na(+) electrochemical potential gradient and the resynthesis of ATP from ADP.

The high-resolution crystal structures and change in enzyme subconformations following binding of cardiotonic steroids to Na,K-ATPase phosphoenzymes for shark Na,K-ATPase in the unbound and ouabain-bound conformation are discussed. Ouabagenin as the most pH-sensitive inhibitor indicated that the steroid hydroxyl side chains are pivotal for observed pH effect, whereas the lactone ring saturation are less important.

The simulations of the equilibrium behavior for different binding models based on thermodynamic data obtained from isothermal titration calorimety is used to explain the large difference in [K.sub.d] values observed in equilibrium and presteady-state kinetic studies due to presence of [Mg.sup.2+] ions. The results from the studies of presteady-state kinetics revealed existence of two ATP binding steps with protein-protein interactions within an [([alpha][beta]).sub.2] diprotomer producing two different ATP binding affinities

The article presents the cholesterol-dependent interaction of polyunsaturated phospholipids with the Na,K-ATPase. The analysis reveals that the phospholipids lead to an increase in the hydrophobic mismatch between lipids and protein in the Na,K-ATPase.