Effect of Fe3+ on Na,K-ATPase: Unexpected activation of ATP hydrolysis

Iron is a key element in cell function; however, its excess in iron overload conditions can be harmful through the generation of reactive oxygen species (ROS) and cell oxidative stress. Activity of Na,K-ATPase has been shown to be implicated in cellular iron uptake and iron modulates the Na,K-ATPase function from different tissues. In this study, we determined the effect of iron overload on Na,K-ATPase activity and established the role that isoforms and conformational states of this enzyme has on this effect. Total blood and membrane preparations from erythrocytes (ghost cells), as well as pig kidney and rat brain cortex, and enterocytes cells (Caco-2) were used. In E1-related subconformations, an enzyme activation effect by iron was observed, and in the E2-related subconformations enzyme inhibition was observed. The enzyme's kinetic parameters were significantly changed only in the Na⁺ curve in ghost cells. In contrast to Na,K-ATPase α2 and α3 isoforms, activation was not observed for the α1 isoform. In Caco-2 cells, which only contain Na,K-ATPase α1 isoform, the FeCl₃ increased the intracellular storage of iron, catalase activity, the production of H₂O₂ and the expression levels of the α1 isoform. In contrast, iron did not affect lipid peroxidation, GSH content, superoxide dismutase and Na,K-ATPase activities. These results suggest that iron itself modulates Na,K-ATPase and that one or more E1-related subconformations seems to be determinant for the sensitivity of iron modulation through a mechanism in which the involvement of the Na, K-ATPase α3 isoform needs to be further investigated.     

Rate determination in phosphorylation of shark rectal Na,K-ATPase by ATP: temperature sensitivity and effects of ADP.

Phosphorylation of shark rectal Na,K-ATPase by ATP in the presence of Na(+) was characterized by chemical quench experiments and by stopped-flow RH421 fluorescence. The appearance of acid-stable phosphoenzyme was faster than the rate of fluorescence increase, suggesting that of the two acid-stable phosphoenzymes formed, RH421 exclusively detects formation of E(2)-P, which follows formation of E(1)-P. The stopped-flow RH421 fluorescence response to ATP phosphorylation was biphasic, with a major fast phase with k(obs) approximately 90 s(-1) and a minor slow phase with a k(obs) of approximately 9 s(-1) (20 degrees C, pH 7.4). The observed rate constants for both the slow and the fast phase could be fitted with identical second-degree functions of the ATP concentration with apparent binding constants of approximately 3.1 x 10(7) M(-1) and 1. 8 x 10(5) M(-1), respectively. Increasing [ADP] decreased k(obs) for the rate of the RH421 fluorescence response to ATP phosphorylation. This could be accounted for by the reaction of ADP with the initially formed E(1)-P followed by a conformational change to E(2)-P. The biphasic stopped-flow RH421 responses to ATP phosphorylation could be simulated, assuming that in the absence of K(+) the highly fluorescent E(2)-P is slowly transformed into the "K(+)-insensitive" E'(2)-P subconformation forming a side branch of the main cycle.     

Involvement of Na/K-ATPase in hydrogen peroxide-induced activation of the Src/ERK pathway in LLC-PK1 cells

We have shown that Na/K-ATPase interacts with Src. Here, we test the role of this interaction in H₂O₂-induced activation of Src and ERK. We found that exposure of LLC-PK1 cells to H₂O₂ generated by the addition of glucose oxidase into the culture medium activated Src and ERK1/2. It also caused a modest reduction in the number of surface Na/K-ATPases and in ouabain-sensitive Rb⁺ uptake. These effects of H₂O₂ seem similar to those induced by ouabain, a specific ligand of Na/K-ATPase, in LLC-PK1 cells. In accordance, we found that the effects of H₂O₂ on Src and ERK1/2 were inhibited in α1 Na/K-ATPase-knockdown PY-17 cells. Whereas expression of wild-type α1 or the A420P mutant α1 defective in Src regulation rescued the pumping activity in PY-17 cells, only α1, and not the A420P mutant, was able to restore the H₂O₂-induced activation of protein kinases. Consistent with this, disrupting the formation of the Na/K-ATPase/Src complex with pNaKtide attenuated the effects of H₂O₂ on the kinases. Moreover, a direct effect of H₂O₂ on Na/K-ATPase-mediated regulation of Src was demonstrated. Finally, H₂O₂ reduced the expression of E-cadherin through the Na/K-ATPase/Src-mediated signaling pathway. Taken together, the data suggest that the Na/K-ATPase/Src complex may serve as one of the receptor mechanisms for H₂O₂ to regulate Src/ERK protein kinases and consequently the phenotype of renal epithelial cells.     

Effects of altering the ATP/ADP ratio on pump-mediated Na/K and Na/Na exchanges in resealed human red blood cell ghosts

Resealed human red blood cell ghosts were prepared to contain a range of ADP concentrations at fixed ATP concentrations and vice versa. ATP/ADP ratios ranging from approximately 0.2 to 50 were set and maintained (for up to 45 min) in this system. ATP and ADP concentrations were controlled by the addition of either a phosphoarginine- or phosphocreatine-based regenerating system. Ouabain-sensitive unidirectional Na efflux was determined in the presence and absence of 15 mM external K as a function of the nucleotide composition. Na/K exchange was found to increase to saturation with ATP (K 1/2 approximately equal to 250 microM), whereas Na/Na exchange (measured in K-free solutions) was a saturating function of ADP (K 1/2 approximately equal to 350 microM). The elevation of ATP from approximately 100 to 1,800 microM did not appreciably affect Na/Na exchange. In the presence of external Na and a saturating concentration of external K, increasing the ADP concentration at constant ATP was found to decrease ouabain-sensitive Na/K exchange. The decreased Na/K exchange that still remained when the ADP/ATP ratio was high was stimulated by removal of external Na. Assuming that under normal substrate conditions the reaction cycle of the Na/K pump is rate-limited by the conformational change associated with the release of occluded K [E2 X (K) X ATP----E1 X ATP + K], increasing ADP inhibits the rate of these transformations by competition with ATP for the E2(K) form. A less likely alternative is that inhibition is due to competition with ATP at the high-affinity site (E1). The acceleration of the Na/K pump that occurs upon removing external Na at high levels of ADP evidently results from a shift in the forward direction of the transformation of the intermediates involved with the release of occluded Na from E1P X (Na). Thus, the nucleotide composition and the Na gradient can modulate the rate at which the Na/K pump operates.     

Hyposmotic stress causes ATP release and stimulates Na,K-ATPase activity in porcine lens

Purinergic receptors in lens epithelium suggest lens function can be altered by chemical signals from aqueous humor or the lens itself. Here we show release of ATP by intact porcine lenses exposed to hyposmotic solution (200 mOsm). 18α-glycyrrhetinic acid (AGA) added together with probenecid eliminated the ATP increase. N-ethylmaleimide (200 μM), an exocytotic inhibitor, had no significant effect on ATP increase. Lenses exposed to hyposmotic solution displayed a ~400% increase of propidium iodide (PI) entry into the epithelium. The increased ability of PI (MW 668) to enter the epithelium suggests possible opening of connexin and/or pannexin hemichannels. This is consistent with detection of connexin 43, connexin 50, and pannexin 1 in the epithelium and the ability of AGA + probenecid to prevent ATP release. Na,K-ATPase activity doubled in the epithelium of lenses exposed to hyposmotic solution. The increase of Na,K-ATPase activity did not occur when apyrase was used to prevent extracellular ATP accumulation or when AGA + probenecid prevented ATP release. The increase of Na,K-ATPase activity was inhibited by the purinergic P2 antagonist reactive blue-2 and pertussis toxin, a G-protein inhibitor, but not by the P2X antagonist PPADS. Hyposmotic solution activated Src family kinase (SFK) in the epithelium, judged by Western blot. The SFK inhibitor PP2 abolished both SFK activation and the Na,K-ATPase activity increase. In summary, hyposmotic shock-induced ATP release is sufficient to activate a purinergic receptor- and SFK-dependent mechanism that stimulates Na,K-ATPase activity. The responses might signify an autoregulatory loop initiated by mechanical stress or osmotic swelling.     

Effect of ADP on Na(+)-Na(+) exchange reaction kinetics of Na,K-ATPase

The whole-cell voltage-clamp technique was used in rat cardiac myocytes to investigate the kinetics of ADP binding to phosphorylated states of Na,K-ATPase and its effects on presteady-state Na(+)-dependent charge movements by this enzyme. Ouabain-sensitive transient currents generated by Na,K-ATPase functioning in electroneutral Na(+)-Na(+) exchange mode were measured at 23 degrees C with pipette ADP concentrations ([ADP]) of up to 4.3 mM and extracellular Na(+) concentrations ([Na](o)) between 36 and 145 mM at membrane potentials (V(M)) from -160 to +80 mV. Analysis of charge-V(M) curves showed that the midpoint potential of charge distribution was shifted toward more positive V(M) both by increasing [ADP] at constant Na(+)(o) and by increasing [Na](o) at constant ADP. The total quantity of mobile charge, on the other hand, was found to be independent of changes in [ADP] or [Na](o). The presence of ADP increased the apparent rate constant for current relaxation at hyperpolarizing V(M) but decreased it at depolarizing V(M) as compared to control (no added ADP), an indication that ADP binding facilitates backward reaction steps during Na(+)-Na(+) exchange while slowing forward reactions. Data analysis using a pseudo three-state model yielded an apparent K(d) of approximately 6 mM for ADP binding to and release from the Na,K-ATPase phosphoenzyme; a value of 130 s(-1) for k(2), a rate constant that groups Na(+) deocclusion/release and the enzyme conformational transition E(1) approximately P --> E(2)-P; a value of 162 s(-1)M(-1) for k(-2), a lumped second-order V(M)-independent rate constant describing the reverse reactions; and a Hill coefficient of approximately 1 for Na(+)(o) binding to E(2)-P. The results are consistent with electroneutral release of ADP before Na(+) is deoccluded and released through an ion well. The same approach can be used to study additional charge-moving reactions and associated electrically silent steps of the Na,K-pump and other transporters.     

Acid-labile ATP and/or ADP/P(i) binding to the tetraprotomeric form of Na/K-ATPase accompanying catalytic phosphorylation-dephosphorylation cycle.

The Na/K-ATPase has been shown to bind 1 and 0.5 mol of (32)P/mol of alpha-chain in the presence [gamma-(32)P]ATP and [alpha-(32)P]ATP, respectively, accompanied by a maximum accumulation of 0.5 mol of ADP-sensitive phosphoenzyme (NaE1P) and potassium-sensitive phosphoenzyme (E2P). The former accumulation was followed by the slow constant liberation of P(i), but the latter was accompanied with a rapid approximately 0.25 mol of acid-labile P(i) burst. The rubidium (potassium congener)-occluded enzyme (approximately 1.7 mol of rubidium/mol of alpha-chain) completely lost rubidium on the addition of sodium + magnesium. Further addition of approximately 100 microM [gamma-(32)P]ATP and [alpha-(32)P]ATP, both induced 0.5 mol of (32)P-ATP binding to the enzyme and caused accumulation of approximately 1 mol of rubidium/mol of alpha-chain, accompanied by a rapid approximately 0.5 mol of P(i) burst with no detectable phosphoenzyme under steady state conditions. Electron microscopy of rotary-shadowed soluble and membrane-bound Na/K-ATPases and an antibody-Na/K-ATPase complex, indicated the presence of tetraprotomeric structures (alphabeta)(4). These and other data suggest that Na/K-ATP hydrolysis occurs via four parallel paths, the sequential appearance of (NaE1P:E.ATP)(2), (E2P:E.ATP:E2P:E. ADP/P(i)), and (KE2:E.ADP/P(i))(2), each of which has been previously referred to as NaE1P, E2P, and KE2, respectively.     

Low affinity superphosphorylation of the Na,K-ATPase by ATP.

Pre-steady-state phosphorylation of purified Na,K-ATPase from red outer medulla of pig kidney was studied at 25 degrees C and an ample range of [tau-32P]ATP concentrations. At 10 microM ATP phosphorylation followed simple exponential kinetics reaching after 40 ms a steady level of 0.76 +/- 0.04 nmol of P/mg of protein with kapp = 73.0 +/- 6.5 s-1. At 500 microM ATP the time course of phosphorylation changed drastically, since the phosphoenzyme reached a level two to four times higher at a much higher rate (kapp greater than or equal to 370 s-1) and in about 40 ms dropped to the same steady level as with 10 microM ATP. This superphosphorylation was not observed in Na,K-ATPase undergoing turnover in a medium with Mg2+, Na+, and ATP, suggesting that it required the enzyme to be at rest. Superphosphorylation depended on Mg2+ and Na+ and was fully inhibited by ouabain and FITC. After denaturation the phosphoenzyme made by superphosphorylation had the electrophoretic mobility of the alpha-subunit of the Na,K-ATPase, and its hydrolysis was accelerated by hydroxylamine. On a molar basis, the stoichiometry of phosphate per ouabain bound was 2.40 +/- 0.60 after phosphorylation with 1000 microM ATP. The results are consistent with the idea that under proper conditions every functional Na,K-ATPase unit can accept two, or more, phosphates of rapid turnover from ATP.     

Control of oxidative phosphorylation by the extramitochondrial ATP/ADP ratio

The control of mitochondrial ATP synthesis by the extramitochondrial adenine nucleotide pattern was investigated with rat liver mitochondria. It is demonstrated that any stationary state between the two limit states of maximum activity (state 3) and of resting activity (state 4) can be obtained by a hexokinase-glucose trap as an ADP-regenerating system. These intermediate states are characterized by stationary respiratory rates, stationary redox levels of the cytochromes b and c and stationary levels of extramitochondrial ATP and ADP between the rates and levels of the limit states. At a constant concentration of inorganic phosphate the activity of mitochondria between the limit states is controlled by the extramitochondrial ATP/ADP ratio independent of the total concentration of adenine nucleotides present. The control range was found to be between ratios of about 5 and 100 at 10 mM phosphate. At lower ratios the mitochondria are in their maximum phosphorylating state. With succinate + rotenone and glutamate + malate the same control range was observed, indicating that it is independent of the nature of substrate oxidized.The results suggest that in the control range the mitochondrial activity is limited by the competition of ADP and ATP for the adenine nucleotide translocator.     

A possible mechanism for the Na,K-ATPase

A model previously described for the Ca²⁺ pump of sarcoplasmic reticulum has been modified in a thought experiment so that it has the properties of a Na,K-adenosinetriphosphatase (ATPase). When the two Ca²⁺-specific sites are changed into three Na⁺-specific sites, and the channel which opens in the actively transporting conformation made univalent- instead of divalent-cation-selective, the model has the properties of the Na-ATPase which is observed on red cell membranes in the absence of both Na⁺ and K⁺ externally. As in the model for the Ca-ATPase the driving force for transport is generated by a change in solvent structure so that a preformed ionic equilibrium is displaced in favour of less-highly hydrated species; in this case highly hydrated Mg²⁺ ions displace the less highly hydrated Na⁺ ions from binding sites; and Na⁺ diffuses out through a simultaneously opened channel. With the addition of three external K⁺-selective sites per α-polypeptide chain, and the constraint that pump units with their external sites occupied by any univalent cation cannot be phosphorylated by ATP, the model turns out to have the properties of a Na,K-ATPase. It operates in the $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ exchange, $\text{Na}{}^{\mathrm{+}}\text{Na}{}^{\mathrm{+}}$ exchange, $\text{K}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ exchange, K⁺-dependent phosphatase, uncoupled Na⁺ efflux and pump reversal modes. It is concluded that if the modified water in the cleft of the phospho-enzymes has properties similar to those of water at 5°C the pump is competent to exchange three intracellular Na⁺ ions for two extracellular K⁺ ions, and one intracellular Na⁺ ion but it is incapable of exchanging three Na⁺ ions for three K⁺ ions.     

Novel regulation of Na, K-ATPase by Src tyrosine kinases in cortical neurons

The Na+, K+-ATPase or Na+, K+-pump plays a critical role in ion homeostasis and many cellular events. The Na+, K+-pump activity is regulated by serine/threonine phosphorylation, the role of tyrosine kinases in the regulation, however, is obscure. We now present novel evidence showing that tyrosine phosphorylation activates the Na+, K+-pump in cortical neurons. The electrogenic activity of the Na+, K+-pump was measured using whole-cell voltage clamp. A tonic activity was revealed by an inward current induced by the specific inhibitor ouabain or strophanthidin; an outward current due to activation of the pump was triggered by raising extracellular K+. The inward and outward currents were attenuated by the tyrosine kinase inhibitor genistein, herbimycin A, or lavendustin A, while blocking tyrosine phosphatases increased the pump current. Down-regulation of the pump current was also seen with the Src inhibitor PP1 and intracellularly applied anti-Lyn or anti-Yes antibody. Consistently, intracellular application of Lyn kinase up-regulated the pump current. Immunoprecipitation and western blotting showed tyrosine phosphorylation and a direct interaction between Lyn and the alpha3 subunit of the Na+, K+-pump. The tyrosine phosphorylation of the alpha3 subunit was reduced by serum deprivation. These data suggest that the Na+, K+-ATPase activity in central neurons is regulated by specific Src tyrosine kinases via a protein-protein mechanism and may play a role in apoptosis.     

In vivo modification of Na,K-ATPase activity in Drosophila

We have constructed and characterized transgenic Drosophila lines with modified Na⁺,K⁺-ATPase activity. Using a temperature dependent promoter from the hsp70 gene to drive expression of wild-type α subunit cDNA, we can conditionally rescue bang-sensitive paralysis and ouabain sensitivity of a Drosophila Na⁺,K⁺-ATPase α subunit hypomorphic mutant, 2206. In contrast, a mutant α subunit (α^D369N) leads to increased bang-sensitive paralysis and ouabain sensitivity. We can also generate temperature dependent phenotypes in wild-type Drosophila using the same hsp70 controlled α transgenes. Ouabain sensitivity was as expected, however, both bang sensitive paralysis or locomotor phenotypes became more severe regardless of the type of α subunit transgene. Using the Gal4-UAS system we have limited expression of α transgenes to cell types that normally express a particular Drosophila Na⁺,K⁺-ATPase β (Nervana) subunit isoform (Nrv1 or 2). The Nrv1-Gal4 driver results in lethality while the Nrv2-Gal4 driver shows reduced viability, locomotor function and uncontrolled wing beating. These transgenic lines will be useful for disrupting function in a broad range of cell types.     

Importance of Na,K-ATPase residue alpha 1-Arg544 in the segment Arg544-Asp567 for high-affinity binding of ATP, ADP, or MgATP

To identify residues involved in ATP binding in the N-domain of the alpha1-subunit of Na,K-ATPase, mutations were directed to the segment Arg(544)-Asp(567), a beta-strand-loop-helix structure with Arg(544) positioned at the mouth of the ATP-binding pocket near the interface to the P-domain. Substitution of Arg(544) with Gln abolished high-affinity binding of free ATP, while substitution with lysine reduced ADP affinity with minor effects on ATP binding. The contribution of Arg(544) to the change in free energy of ATP binding was estimated to 6.9 kJ/mol (DeltaDeltaG(b)) from double mutations with Asp(369) and to 7.8 kJ/mol from the MgATP dependence of phosphorylation. The phosphorylation data show that binding of Mg(2+) may increase the apparent affinity of wild-type enzyme for ATP [K(1/2)(ATP) 12 nM]. Moderately reduced affinities for ATP were seen after mutations of Asp(555), Glu(556), Asp(565), or Asp(567) with DeltaDeltaG(b) approximately equals 0.5-3 kJ/mol. Mutations of Cys(549) did not affect ATP binding. In conclusion, Arg(544) is important for binding of ATP or ADP, probably by stabilizing the beta- or gamma-phosphate moieties and aligning the gamma-phosphate for interaction with the carboxylate group of Asp(369).     

Digitalis-induced cell signaling by the sodium pump: On the relation of Src to Na/K-ATPase

In addition to performing its essential transport function, the sodium pump also activates multiple cell signaling pathways in response to digitalis drugs such as ouabain. Based mainly on cell-free studies with mixtures of purified Src kinase and Na⁺/K⁺-ATPase, a well-advocated hypothesis on how ouabain initiates the activation of signaling pathways is that there is a preexisting physiological complex of inactive Src bound to the α-subunit of Na⁺/K⁺-ATPase, and that ouabain binding to this subunit disrupts the bound Src and activates it. Because of the published disagreements of the results of such cell-free experiments of two other laboratories, our aim was to attempt the resolution of these discrepancies. We reexamined the effects of ouabain, vanadate, and oligomycin on mixtures of Src, Na⁺/K⁺-ATPase, Mg²⁺, and ATP as specified in prior studies; and assayed for Src-418 autophosphorylation as the measure of Src activation. In contrast to the findings of the proponents of the above hypothesis, our results showed similar effects of the three inhibitors of Na⁺/K⁺-ATPase; indicating that Src activation in such experiments is primarily due to the ATP-sparing effect of the ATPase inhibitor on the mixture of two enzymes competing for ATP. We conclude that there is no solid evidence for direct molecular interaction of Src with Na⁺/K⁺-ATPase under physiological conditions.     

New evidence for ATP binding induced catalytic subunit interactions in pig kidney Na/K-ATPase

Pig kidney Na/K-ATPase preparations showed a positive cooperative effect for pNPP in Na-pNPPase activity. Measurements of the Na-pNPPase activity, Na-ATPase activity and the accumulation of phosphoenzyme (EP) under conditions of pNPP saturation showed several different ATP affinities. The presence of pNPP reduced both the maximum amount of EP and Na-ATPase activity to half showing a value of 4 and a 3,700-fold reduced ATP affinity for EP formation, and a 7 and 1,300-fold reduced affinity for Na-ATPase activity. The presence of low concentrations of ATP in the phosphorylation induced a 2-fold enhancement in Na-pNPPase activity despite a reduction in available pNPP sites. However, higher concentrations of ATP inhibited the Na-pNPPase activity and a much higher concentration of ATP increased both the phosphorylation and Na-ATPase activity to the maximum levels. The maximum Na-pNPPase activity was 1.7 and 3.4-fold higher without and with ATP, respectively, than the maximum Na-ATPase activity. These data and the pNPP dependent reduction in both Na-ATPase activity and the amount of enzyme bound ATP provide new evidence to show that ATP, pNPP and ATP with pNPP, respectively, induce different subunit interactions resulting a difference in the maximum Na(+)-dependent catalytic activity in tetraprotomeric Na/K-ATPase.     

Mechanism of brain Na,K-ATPase activation by adrenaline]

Norepinephrine stimulates Na, K-ATPase from rat brain homogenates at concentrations of 10(-4)--10(-5) and 10(-7)--10(-8) M. A low concentration maximum is observed after 48 hrs of incubation at -20 degrees C and is not changed by the addition of alpha-tocopherol, glycerol and MAO inhibitor ipraside. The maximum observed at the mediator concentration equal to 10(-4)--10(-5) M is eliminated after treatment with EGTA. At all concentrations of norepinephrine the enzyme stimulation is removed by the alpha-adrenoblocker phentolamine. The activated enzyme reveals lower sensitivity to Ca2+ induced inhibition. The role of Ca2+ and conformational state of the membranes in the realization of the remote effect on the adrenoreceptor-Na, K-ATPase system is discussed.     

Interaction of Na,K-ATPase with modifying ATP analogs and chloromethylphosphonic acid

The interaction of synthetic ATP analogs, containing active groups in the triphosphate moiety and in the 8-position of the nucleotide molecule, with highly purified Na, K-ATPase from the medullar layer of porcine kidney was studied. It was found that 11 out of 17 ATP analogs studied irreversibly inhibit the ATPase activity of the enzyme. The pH optimum of the enzyme inactivation by adenosine-5'-(beta-chloroethylphosphate) and adenosine-5'-(p-fluorosulfonylphenylphosphate) beside the pronounced protective effect of ATP suggests possible covalent blocking of histidine and dicarboxylic amino acid residues in the enzyme active center. The irreversible inhibition of the enzyme by "oxo-ATP" containing aldehyde groups in the modified ribose residue in the presence of sodium borohydride suggests a possible presence of the lysine residue epsilon-amino group in the ATP binding site of the enzyme. Na, K-ATPase was found to possess an inorganic phosphate binding site, which is specifically blocked by chloromethylphosphonic acid. the accessibility of this site for modification depends on ATP, NA+ and K+.     

ATP and magnesium drive conformational changes of the Na/K-ATPase cytoplasmic headpiece

Conformational changes of the Na⁺/K⁺-ATPase isolated large cytoplasmic segment connecting transmembrane helices M4 and M5 (C45) induced by the interaction with enzyme ligands (i.e. Mg²⁺ and/or ATP) were investigated by means of the intrinsic tryptophan fluorescence measurement and molecular dynamic simulations. Our data revealed that this model system consisting of only two domains retained the ability to adopt open or closed conformation, i.e. behavior, which is expected from the crystal structures of relative Ca²⁺-ATPase from sarco(endo)plasmic reticulum for the corresponding part of the entire enzyme. Our data revealed that the C45 is found in the closed conformation in the absence of any ligand, in the presence of Mg²⁺ only, or in the simultaneous presence of Mg²⁺ and ATP. Binding of the ATP alone (i.e. in the absence of Mg²⁺) induced open conformation of the C45. The fact that the transmembrane part of the enzyme was absent in our experiments suggested that the observed conformational changes are consequences only of the interaction with ATP or Mg²⁺ and may not be related to the transported cations binding/release, as generally believed. Our data are consistent with the model, where ATP binding to the low-affinity site induces conformational change of the cytoplasmic part of the enzyme, traditionally attributed to E2 → E1 transition, and subsequent Mg²⁺ binding to the enzyme-ATP complex induces in turn conformational change traditionally attributed to E1 → E2 transition.