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Fast changes in environmental oxygen availability translate into shifts in mitochondrial free radical production. An increase in intraerythrocytic reduced glutathione (GSH) during deoxygenation would support the detoxification of exogenous oxidants released into the circulation from hypoxic peripheral tissues. Although reported, the mechanism behind this acute oxygen-dependent regulation of GSH in red blood cells remains unknown. This study explores the role of hemoglobin (Hb) in the oxygen-dependent modulation of GSH levels in red blood cells. We have demonstrated that a decrease in Hb O

Background S-glutathionylation is the formation of disulfide bonds between the tripeptide glutathione and cysteine residues of the protein, protecting them from irreversible oxidation and in some cases causing change in their functions. Regulatory glutathionylation of proteins is a controllable and reversible process associated with cell response to the changing redox status. Prediction of cysteine residues that undergo glutathionylation allows us to find new target proteins, which function can be altered in pathologies associated with impaired redox status. We set out to analyze this issue and create new tool for predicting S-glutathionylated cysteine residues. Results One hundred forty proteins with experimentally proven S-glutathionylated cysteine residues were found in the literature and the RedoxDB database. These proteins contain 1018 non-S-glutathionylated cysteines and 235 S-glutathionylated ones. Based on 235 S-glutathionylated cysteines, non-redundant positive dataset of 221 heptapeptide sequences of S-glutathionylated cysteines was made. Based on 221 heptapeptide sequences, a position-specific matrix was created by analyzing the protein sequence near the cysteine residue (three amino acid residues before and three after the cysteine). We propose the method for calculating the glutathionylation propensity score, which utilizes the position-specific matrix and a criterion for predicting glutathionylated peptides. Conclusion Non-S-glutathionylated sites were enriched by cysteines in − 3 and + 3 positions. The proposed prediction method demonstrates 76.6% of correct predictions of S-glutathionylated cysteines. This method can be used for detecting new glutathionylation sites, especially in proteins with an unknown structure.

The functioning of the N, K-ATPase depends on the redox status of cells and its activity is inhibited by oxidative stress and hypoxia. We previously found that redox sensitivity of the Na,K-ATPase is mediated by glutathionylation of the α-subunit. An increase in the level of glutathionylation of cysteine residues in the Na,K-ATPase α-subunit under stressful conditions leads to a decrease in the activity of the enzyme and a change in its receptor function. The structure of the Na,K-ATPase undergoes significant conformational changes during functioning. The effects of enzyme conformation on its ability to undergo glutathionylation are not clear. Here we show that the highest level of glutathionylation in the α-subunit of Na,K-ATPase is achieved in the E1 (Na+-induced) conformation. The transition of the Na,K-ATPase to the E2 (K+-induced) conformation leads to a decrease in the efficiency of glutathionylation. The lowest efficiency of Na,K-ATPase glutathionylation was observed in the E2P and E2P ouabain states. According to molecular modelling data, the maximum number of cysteine residues available for glutathionylation are present in the E1P conformation. In the E2P conformation, the main functional cysteine residues (Cys204, Cys242, Cys452, and Cys456) are buried from the solvent, which makes them inaccessible for glutathionylation. Thus, the efficiency of α-subunit glutathionylation depends on enzyme conformation, which is altered by bound ligands and proteins. A shift in the E1/E2 equilibrium towards prevalence of E1 can lead to better access for the relevant ligands and proteins to the binding site located in the Na,K-ATPase α-subunit. Na,K-ATPase.

We have shown that the inhibition of Na,K-ATPase during its long-term incubation with amyloid beta (Aβ42), an Alzheimer’s disease protein, is caused by the change in the thiol redox status of cells leading to induction of glutathionylation α-subunit of Na,K-ATPase. To restore the activity of Na,K-ATPase, it is proposed to use reducing agents, which promote normalization of the redox status of cells and deglutathionylation of the protein.

It was shown earlier that a 67-kDa protein purified from mouse kidney using polyclonal antibodies against melittin (a peptide from bee venom) interacted with Na,K-ATPase from rabbit kidney. In this study, a 43-kDa proteolytic fragment of Na,K-ATPase α-subunit interacting with the 67-kDa melittin-like protein was found. The α-subunit was hydrolyzed by trypsin in the presence of 0.5 mM ouabain (E2-conformation of Na,K-ATPase). A proteolytic fragment interacting with the 67-kDa melittin-like protein that was identified by mass-spectrometry is a region of the cytoplasmic domain of Na,K-ATPase α-subunit located between amino acid residues 591 and 775. The fragment includes a conservative DPPRA motif that occurs in many P-type ATPases. It was shown earlier that this motif of H,K-ATPase from gastric mucosa binds to melittin. We suggest that namely this motif of P-type ATPases is able to interact with proteins containing melittin-like modules.

Many viruses induce oxidative stress and cause S-glutathionylation of Cys residues of the host and viral proteins. Changes in cell functioning during viral infection may be associated with glutathionylation of a number of key proteins including Na,K-ATPase which creates a gradient of sodium and potassium ions. It was found that Na,K-ATPaseα-subunit has a basal glutathionylation which is not abrogated by reducing agent. We have shown that acute hypoxia leads to increase of total glutathionylation level of Na,K-ATPaseα-subunit; however, basal glutathionylation ofα-subunit increases under prolonged hypoxia only. The role of basal glutathionylation in Na,K-ATPase function remains unclear. Understanding significance of basal glutathionylation is complicated by the fact that there are no X-ray structures of Na,K-ATPase with the identified glutathione molecules. We have analyzed all X-ray structures of the Na,K-ATPaseα-subunit from pig kidney and found that there are a number of isolated cavities with unresolved electron density close to the relevant cysteine residues. Analysis of the structures showed that this unresolved density in the structure can be occupied by glutathione associated with cysteine residues. Here, we discuss the role of basal glutathionylation of Na,K-ATPaseα-subunit and provide evidence supporting the view that this modification is cotranslational.

We found earlier that Na,K-ATPase is purified from duck salt glands in partially glutathionylated state (up to 13 of the 23 cysteine residues of the Na,K-ATPase catalytic α-subunit can be S-glutathionylated). To determine the effect of glutathionylation on the enzyme conformation, we have analyzed the products of trypsinolysis of Na,K-ATPase α-subunit in different conformations with different extent of glutathionylation. Incubation of the protein in the E1 conformation with trypsin produced a large fragment with a molecular mass (MM) of 80 kDa with the following formation of smaller fragments with MM 40, 35.5, and 23 kDa. Tryptic digestion of Na,K-ATPase in the E2 conformation also resulted in the generation of the fragments with MM 40, 35.5, and 23 kDa. Deglutathionylation of Na,K-ATPase α-subunit increases the rate of proteolysis of the enzyme in both E1 and E2 conformations. The pattern of tryptic digestion of the α-subunit in E2 conformation additionally glutathionylated with oxidized glutathione is similar to that of partially deglutathionylated Na,K-ATPase. The pattern of tryptic digestion of the additionally glutathionylated α-subunit in E1 conformation is similar to that of the native enzyme. The highest rate of trypsinolysis was observed for the α-subunit in complex with ouabain (E2-OBN conformation). Additional glutathionylation increased the content of high-molecular-weight fragments among the digestion products, as compared to the native and deglutathionylated enzymes. The data obtained were confirmed using molecular mod-eling that revealed that number of sites accessible for trypsinolysis is higher in the E2P-OBN conformation than in the E1-and E2-conformations and that glutathionylation decreases the number of sites accessible for trypsin. Therefore, glu-tathionylation affects enzyme conformation and its sensitivity to trypsinolysis. The mechanisms responsible for the changes in the Na,K-ATPase sensitivity to trypsinolysis depending on the level of enzyme glutathionylation and increase in the enzyme sensitivity to proteolysis upon its binding to ouabain, as well as physiological role of these phenomena, are discussed.

Our previous findings suggested that reversible thiol modifications of cysteine residues within the actuator (AD) and nucleotide binding domain (NBD) of the Na,K-ATPase may represent a powerful regulatory mechanism conveying redox- and oxygen-sensitivity of this multifunctional enzyme. S-glutathionylation of Cys244 in the AD and Cys 454-458-459 in the NBD inhibited the enzyme and protected cysteines’ thiol groups from irreversible oxidation under hypoxic conditions. In this study mutagenesis approach was used to assess the role these cysteines play in regulation of the Na,K-ATPase hydrolytic and signaling functions. Several constructs of mouse α1 subunit of the Na,K-ATPase were produced in which Cys244, Cys 454-458-459 or Cys 244-454-458-459 were replaced by alanine. These constructs were expressed in human HEK293 cells. Non-transfected cells and those expressing murine α1 subunit were exposed to hypoxia or treated with oxidized glutathione (GSSG). Both conditions induced inhibition of the wild type Na,K-ATPase. Enzymes containing mutated mouse α1 lacking Cys244 or all four cysteines (Cys 244-454-458-459) were insensitive to hypoxia. Inhibitory effect of GSSG was observed for wild type murine Na,K-ATPase, but was less pronounced in Cys454-458-459Ala mutant and completely absent in the Cys244Ala and Cys 244-454-458-459Ala mutants. In cells, expressing wild type enzyme, ouabain induced activation of Src and Erk kinases under normoxic conditions, whereas under hypoxic conditions this effect was inversed. Cys454-458-459Ala substitution abolished Src kinase activation in response to ouabain treatment, uncoupled Src from Erk signaling, and interfered with O2-sensitivity of Na,K-ATPase signaling function. Moreover, modeling predicted that S-glutathionylation of Cys 458 and 459 should prevent inhibitory binding of Src to NBD. Our data indicate for the first time that cysteine residues within the AD and NBD influence hydrolytic as well as receptor function of the Na,K-ATPase and alter responses of the enzyme to hypoxia or upon treatment with cardiotonic steroids., Graphical abstract fx1

Sodium-potassium adenosine triphosphatase (Na,K-ATPase) creates a gradient of sodium and potassium ions necessary for the viability of animal cells, and it is extremely sensitive to intracellular redox status. Earlier we found that regulatory glutathionylation determines Na,K-ATPase redox sensitivity but the role of basal glutathionylation and other redox modifications of cysteine residues is not clear. The purpose of this study was to detect oxidized, nitrosylated, or glutathionylated cysteine residues in Na,K-ATPase, evaluate the possibility of removing these modifications and assess their influence on the enzyme activity. To this aim, we have detected such modifications in the Na,K-ATPase α1-subunit purified from duck salt glands and tried to eliminate them by chemical reducing agents and the glutaredoxin1/glutathione reductase enzyme system. Detection of cysteine modifications was performed using mass spectrometry and Western blot analysis. We have found that purified Na,K-ATPase α1-subunit contains glutathionylated, nitrosylated, and oxidized cysteines. Chemical reducing agents partially eliminate these modifications that leads to the slight increase of the enzyme activity. Enzyme system glutaredoxin/glutathione reductase, unlike chemical reducing agents, produces significant increase of the enzyme activity. At the same time, the enzyme system deglutathionylates native Na,K-ATPase to a lesser degree than chemical reducing agents. This suggests that the enzymatic reducing system glutaredoxin/glutathione reductase specifically affects glutathionylation of the regulatory cysteine residues of Na,K-ATPase α1-subunit.

By maintaining the Na+ and K+ transmembrane gradient mammalian Na,K-ATPase acts as a key regulator of neuronal electrotonic properties. Na,K-ATPase has an important role in synaptic transmission and memory formation. Accumulation of beta-amyloid (Aβ) at the early stages of Alzheimer’s disease is accompanied by reduction of Na,K-ATPase functional activity. The molecular mechanism behind this phenomenon is not known. Here we show that the monomeric Aβ(1-42) forms a tight (Kd of 3 μM), enthalpy-driven equimolar complex with α1β1 Na,K-ATPase. The complex formation results in dose-dependent inhibition of the enzyme hydrolytic activity. The binding site of Aβ(1-42) is localized in the “gap” between the alpha- and beta-subunits of Na,K-ATPase, disrupting the enzyme functionality by preventing the subunits from shifting towards each other. Interaction of Na,K-ATPase with exogenous Aβ(1-42) leads to a pronounced decrease of the enzyme transport and hydrolytic activity and Src-kinase activation in neuroblastoma cells SH-SY5Y. This interaction allows regulation of Na,K-ATPase activity by short-term increase of the Aβ(1-42) level. However prolonged increase of Aβ(1-42) level under pathological conditions could lead to chronical inhibition of Na,K-ATPase and disruption of neuronal function. Taken together, our data suggest the role of beta-amyloid as a novel physiological regulator of Na,K-ATPase.

Control over the Na,K-ATPase function plays a central role in adaptation of the organisms to hypoxic and anoxic conditions. As the enzyme itself does not possess O2 binding sites its "oxygen-sensitivity" is mediated by a variety of redox-sensitive modifications including S-glutathionylation, S-nitrosylation, and redox-sensitive phosphorylation. This is an overview of the current knowledge on the plethora of molecular mechanisms tuning the activity of the ATP-consuming Na,K-ATPase to the cellular metabolic activity. Recent findings suggest that oxygen-derived free radicals and H2O2, NO, and oxidized glutathione are the signaling messengers that make the Na,K-ATPase "oxygen-sensitive." This very ancient signaling pathway targeting thiols of all three subunits of the Na,K-ATPase as well as redox-sensitive kinases sustains the enzyme activity at the "optimal" level avoiding terminal ATP depletion and maintaining the transmembrane ion gradients in cells of anoxia-tolerant species. We acknowledge the complexity of the underlying processes as we characterize the sources of reactive oxygen and nitrogen species production in hypoxic cells, and identify their targets, the reactive thiol groups which, upon modification, impact the enzyme activity. Structured accordingly, this review presents a summary on (i) the sources of free radical production in hypoxic cells, (ii) localization of regulatory thiols within the Na,K-ATPase and the role reversible thiol modifications play in responses of the enzyme to a variety of stimuli (hypoxia, receptors' activation) (iii) redox-sensitive regulatory phosphorylation, and (iv) the role of fine modulation of the Na,K-ATPase function in survival success under hypoxic conditions. The co-authors attempted to cover all the contradictions and standing hypotheses in the field and propose the possible future developments in this dynamic area of research, the importance of which is hard to overestimate. Better understanding of the processes underlying successful adaptation strategies will make it possible to harness them and use for treatment of patients with stroke and myocardial infarction, sleep apnoea and high altitude pulmonary oedema, and those undergoing surgical interventions associated with the interruption of blood perfusion., The review was funded by the grants of Swiss Nationa lScience Foundation IZK0Z3_157269/1 and 310030_124970/1 to AB and Russian Science Foundation (Grant#14-14-01152) to IP Spanish Government grants (partially funded by the European Union FEDER/EDRF) PI12/00875 and PI15/00107 and a grant from the Fundación Domingo Martínez are supporting AM, PH received a travel grant from the Instituto de Investigación Sanitaria Princesa (to PH),and by COST actions TD0901 (HypoxiaNet) and CM1001. PH is recipient of a pre-doctoral FPU fellowship from the Spanish Government and AM is supported by the I3SNS programme (ISCIII, Spanish Government, partially funded by FEDER/ERDF)

Bacterial RNases are promising tools for the development of anticancer drugs. Neoplastic transformation leads to enhanced accumulation of rRNA and tRNA, and altered expression of regulatory noncoding RNAs. Cleavage of RNA in cancer cells is the main reason for the cytotoxic effects of exogenic RNases. We have shown that binase, a cytotoxic ribonuclease from Bacillus intermedius, affects the total amount of intracellular RNA and the expression of proapoptotic and antiapoptotic mRNAs. For four cell lines, we visualized cellular RNA by fluorescence microscopy, and determined RNA levels, viability and apoptosis by flow cytometry. We found that the level of cellular RNA was decreased in cells that were sensitive to the cytotoxic effects of binase. The RNA level was lowered by 44% in HEK cells transfected with the hSK4 gene of the Ca2+-activated potassium channels (HEKhSK4) and by 20% in kit-transformed myeloid progenitor FDC-P1iR1171 cells. The most significant decrease in RNA levels was registered in the subpopulations of apoptotic cells. However, the binase-induced RNA decrease did not correlate with apoptosis. Kit-transformed cells with binase-induced RNA decrease retained viability if the interleukin-dependent proliferation pathway was activated. Using quantitative RT-PCR with RNA samples isolated from the binase-treated HEKhSK4 cells, we found that the amount of mRNA of the antiapoptotic bcl-2 gene in vivo was reduced about two-fold. In contrast, expression of the proapoptotic genes p53 and hSK4 was increased 1.5-fold and 4.3-fold, respectively. These results show that binase is a regulator of RNA-dependent processes of cell proliferation and apoptosis.

Adjustment of the Na/K ATPase activity to changes in oxygen availability is a matter of survival for neuronal cells. We have used freshly isolated rat cerebellar granule cells to study oxygen sensitivity of the Na/K ATPase function. Along with transport and hydrolytic activity of the enzyme we have monitored alterations in free radical production, cellular reduced glutathione, and ATP levels. Both active K+ influx and ouabain-sensitive inorganic phosphate production were maximal within the physiological pO2 range of 3–5 kPa. Transport and hydrolytic activity of the Na/K ATPase was equally suppressed under hypoxic and hyperoxic conditions. The ATPase response to changes in oxygenation was isoform specific and limited to the α1-containing isozyme whereas α2/3-containing isozymes were oxygen insensitive. Rapid activation of the enzyme within a narrow window of oxygen concentrations did not correlate with alterations in the cellular ATP content or substantial shifts in redox potential but was completely abolished when NO production by the cells was blocked by l-NAME. Taken together our observations suggest that NO and its derivatives are involved in maintenance of high Na/K ATPase activity under physiological conditions.

Ion pump, Na,K-ATPase specifically binds cardiotonic steroids (CTS), which leads to inhibition of the enzyme activity and activation of signaling network in the cell. We have studied interaction of Na,K-ATPase with CTS of two different types – marinobufagenin and ouabain. We have shown that both CTS inhibit activity of Na,K-ATPase with the same Ki values, but binding of ouabain is sensitive to the conformation of Na,K-ATPase while binding of marinobufagenin is not. Furthermore, binding of ouabain and marinobufagenin results in different structural changes in Na,K-ATPase. Our data allow to explain the diversity of effects on the receptor function of Na,K-ATPase caused by different types of CTS.

GTP hydrolysis catalyzed in the ribosome by a complex of two polypeptide release factors, eRF1 and eRF3, is required for fast and efficient termination of translation in eukaryotes. Here, isothermal titration calorimetry is used for the quantitative thermodynamic characterization of eRF3 interactions with guanine nucleotides, eRF1 and Mg2+. We show that (i) eRF3 binds GDP (K(d) = 1.9 microM) and this interaction depends only minimally on the Mg(2+) concentration; (ii) GTP binds to eRF3 (K(d) = 0.5 microM) only in the presence of eRF1 and this interaction depends on the Mg2+ concentration; (iii) GTP displaces GDP from the eRF1*eRF3*GDP complex, and vice versa; (iv) eRF3 in the GDP-bound form improves its ability to bind eRF1; (v) the eRF1*eRF3 complex binds GDP as efficiently as free eRF3; (vi) the eRF1*eRF3 complex is efficiently formed in the absence of GDP/GTP but requires the presence of the C-terminus of eRF1 for complex formation. Our results show that eRF1 mediates GDP/GTP displacement on eRF3. We suggest that after formation of eRF1*eRF3*GTP*Mg2+, this quaternary complex binds to the ribosomal pretermination complex containing P-site-bound peptidyl-tRNA and the A-site-bound stop codon. The guanine nucleotide binding properties of eRF3 and of the eRF3*eRF1 complex profoundly differ from those of prokaryotic RF3.

The Na/K ATPase, or Na/K pump, is an enzyme converting chemical energy of ATP hydrolysis into the energy of electrochemical sodium and potassium gradient that is used to maintain cellular volume, pH and Ca2+ levels. Apart from maintaining the cellular Na+ and K+ concentrations, Na/K ATPase is involved in signal transduction including activation of mitogenic and proliferative signalling cascades. Changes in the Na/K ATPase activity have an impact on cellular metabolic and redox state, since it controls mitochondrial reactive oxygen species (ROS) levels and ATP utilisation rates. Na/K ATPase is known to be oxygenand redoxsensitive. Alterations in the Na/K pump activity in response to hypoxia-reoxygenation or shifts in cellular and environmental redox state play an important role in pathological and adaptive responses of cells to hypoxia or ischemia. Despite intensive studies, the mechanisms involved in redoxand oxygen-induced regulation of the Na/K ATPase remain largely unknown. The present review focuses on the possible targets of action of oxygen, oxidants and reductants on the Na/K ATPase and mechanisms of oxygenand redox-sensitivity of the enzyme at the molecular and cellular levels. Finally, we also consider factors involved in systemic responses to hypoxia that influence activity of the transporter as well as potential physiological and pathological outcome

Active transport of sodium and potassium ions by Na,K-ATPase is accompanied by the enzyme conformational transition between E1 and E2 states. ATP and ADP bind to Na,K-ATPase in the E1 conformation with similar affinity but the properties of enzyme in complexes with these nucleotides are different. We have studied thermodynamics of Na,K-ATPase binding with adenine nucleotides at different temperatures using isothermal titration calorimetry. Our data indicate that β-phosphate is involved in complex formation by increasing the affinity of adenine nucleotides to Na,K-ATPase by an order of magnitude, while γ-phosphate does not affect it. ATP binding to Na,K-ATPase in contrast to ADP binding generates a structural transition in the enzyme, which is consistent with the movement of a significant portion of the surface area to a solvent-protected state. We propose that ATP binding leads to convergence of the nucleotide-binding and phosphorylation domains transferring the enzyme from the "E1-open" to "E1-closed" conformation ready for phosphorylation.

Translational GTPases (trGTPases) are involved in all four stages of protein biosynthesis: initiation, elongation, termination and ribosome recycling. The trGTPases Initiation Factor 2 (IF2) and Elongation Factor G (EF-G) respectively orchestrate initiation complex formation and translocation of the peptidyl-tRNA:mRNA complex through the bacterial ribosome. The ribosome regulates the GTPase cycle and efficiently discriminates between the GDP- and GTP-bound forms of these proteins. Using Isothermal Titration Calorimetry, we have investigated interactions of IF2 and EF-G with the sarcin-ricin loop of the 23S rRNA, a crucial element of the GTPase-associated center of the ribosome. We show that binding of IF2 and EF-G to a 27 nucleotide RNA fragment mimicking the sarcin-ricin loop is mutually exclusive with that of GDP, but not of GTP, providing a mechanism for destabilization of the ribosome-bound GDP forms of translational GTPases.

Na,K-ATPase is highly sensitive to changes in the redox state, and yet the mechanisms of its redox sensitivity remain unclear. We have explored the possible involvement of S-glutathionylation of the catalytic α subunit in redox-induced responses. For the first time, the presence of S-glutathionylated cysteine residues was shown in the α subunit in duck salt glands, rabbit kidneys, and rat myocardium. Exposure of the Na,K-ATPase to oxidized glutathione (GSSG) resulted in an increase in the number of S-glutathionylated cysteine residues. Increase in S-glutathionylation was associated with dose- and time-dependent suppression of the enzyme function up to its complete inhibition. The enzyme inhibition concurred with S-glutathionylation of the Cys-454, -458, -459, and -244. Upon binding of glutathione to these cysteines, the enzyme was unable to interact with adenine nucleotides. Inhibition of the Na,K-ATPase by GSSG did not occur in the presence of ATP at concentrations above 0.5 mm. Deglutathionylation of the α subunit catalyzed by glutaredoxin or dithiothreitol resulted in restoration of the Na,K-ATPase activity. Oxidation of regulatory cysteines made them inaccessible for glutathionylation but had no profound effect on the enzyme activity. Regulatory S-glutathionylation of the α subunit was induced in rat myocardium in response to hypoxia and was associated with oxidative stress and ATP depletion. S-Glutathionylation was followed by suppression of the Na,K-ATPase activity. The rat α2 isoform was more sensitive to GSSG than the α1 isoform. Our findings imply that regulatory S-glutathionylation of the catalytic subunit plays a key role in the redox-induced regulation of Na,K-ATPase activity.

Microbial RNases along with such animal RNases as onconase and BS-RNase are a promising basis for developing new antitumor drugs. We have shown that the Bacillus intermedius RNase (binase) induces selective apoptosis of transformed myeloid cells. It attacks artificially expressing activated c-Kit myeloid progenitor FDC cells and chronic myelogenous leukemia cells K562. Binase did not induce apoptosis in leukocytes of healthy donors and in normal myeloid progenitor cells. The inability of binase to initiate expression of activation markers CD69 and IFN-gamma in CD4+ and CD8+ T-lymphocytes testifies that enzyme is devoid of superantigenic properties. Altogether, these results demonstrate that binase possesses therapeutic opportunities for treatment of genotyped human neoplasms expressing activated kit.

Reduction in ATP generation under conditions of limited oxygen supply triggers acute decrease in ATP consumption by turning off the Na,K-pump. This adaptive response is effective when followed by “channel arrest”, but may be lethal when not associated with reduction in passive Na and K permeability. Oxygen sensing by the Na,K-ATPase is mediated by way of reversible thiol modifications. We have identified the four regulatory cysteine residues within the ATP binding site of the catalytic α subunit that, when S-glutathionylated, prevent binding of ATP to the enzyme. S-glutathionylation may only occur under conditions of mild ATP depletion associated with GSSG accumulation in the enzyme proximity. Identification of the residues was performed by mass spectrometry on purified Na,K-ATPase which was completely inactivated by treatment with GSSG. In silico modelling and titration experiments using isothermal scanning calorimetry confirmed competition of GSSG and ATP for the ATP binding domain. Finally, a set of HEK293 cell lines transfected with murine α1 subunit protein with point mutations of the candidate cysteines for alanines was produced and the oxygen-insensitivity of mutant murine Na,K-ATPase was proven. The lack of oxygen-sensitive made the transfected host cells less hypoxia-tolerant. We furthermore advanced in identification of the source of hypoxia-induced free radical production triggering GSSG production which is required for making the Na,K-ATPase oxygen-sensitive. A burst of superoxide anion production originating from the mitochondria was observed in several cell types including cerebellar granule cells during the first 10-15 minutes of hypoxia. The latter could be inhibited by silencing or pharmacological inhibition of the mitochondrial Na/Ca exchanger by CGP-37157. Administration of CGP-37157 preserved the Na,K-ATPase function in hypoxic cerebellar granule cells, decreased the intracellular GSSG levels and reduced S-glutathionylation of the α subunit.

Redox-induced regulation of the Na-K-ATPase was studied in dispersed rat cerebellar granule cells. Intracellular thiol redox state was modulated using glutathione (GSH)-conjugating agents and membrane-permeable ethyl ester of GSH (et-GSH) and Na-K-ATPase transport and hydrolytic activity monitored as a function of intracellular reduced thiol concentration. Depletion of cytosolic and mitochondrial GSH pools caused an increase in free radical production in mitochondria and rapid ATP deprivation with a subsequent decrease in transport but not hydrolytic activity of the Na-K-ATPase. Selective conjugation of cytosolic GSH did not affect free radical production and Na-K-ATPase function. Unexpectedly, overloading of cerebellar granule cells with GSH triggered global free radical burst originating most probably from GSH autooxidation. The latter was not followed by ATP depletion but resulted in suppression of active K+influx and a modest increase in mortality. Suppression of transport activity of the Na-K-ATPase was observed in granule cells exposed to both permeable et-GSH and impermeable GSH, with inhibitory effects of external and cytosolic GSH being additive. The obtained data indicate that redox state is a potent regulator of the Na-K-ATPase function. Shifts from an “optimal redox potential range” to higher or lower levels cause suppression of the Na-K pump activity.

Isomerization of Asp7 leads to increased toxic effect of amyloid-β42 on human neuronal cells