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 ∼5-6 nm³ (i.e., ∼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 nm³. Analysis of 15 crystal structures of the homologous Ca-ATPase reveals an increase in PEG-inaccessible water space of ∼22 nm³ between the E₁-nucleotide bound forms and the E₂-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 E₁ and E₂ conformations of the Ca-ATPase.     

Mechanism of the Na,K-ATPase Inhibition by MCS Derivatives

The previously reported class of potent inorganic inhibitors of Na,K-ATPase, named MCS factors, was shown to inhibit not only Na,K-ATPase but several P-type ATPases with high potency in the sub-micromolar range. These MCS factors were found to bind to the intracellular side of the Na, K-ATPase. The inhibition is not competitive with ouabain binding, thus excluding its role as cardiac-steroid-like inhibitor of the Na,K-ATPase. The mechanism of inhibition of Na,K-ATPase was investigated with the fluorescent styryl dye RH421, a dye known to report changes of local electric fields in the membrane dielectric. MCS factors interact with the Na,K-ATPase in the E₁ conformation of the ion pump and induce a conformational rearrangement that causes a change of the equilibrium dissociation constant for one of the first two intracellular cation binding sites. The MCS-inhibited state was found to have bound one cation (H⁺, Na⁺ or K⁺) in one of the two unspecific binding sites, and at high Na⁺ concentrations another Na⁺ ion was bound to the highly Na⁺-selective ion-binding site.     

The human non-gastric H,K-ATPase has a different cation specificity than the rat enzyme

The primary sequence of non-gastric H,K-ATPase differs much more between species than that of Na,K-ATPase or gastric H,K-ATPase. To investigate whether this causes species-dependent differences in enzymatic properties, we co-expressed the catalytic subunit of human non-gastric H,K-ATPase in Sf9 cells with the β₁ subunit of rat Na,K-ATPase and compared its properties with those of the rat enzyme (Swarts et al., J. Biol. Chem. 280, 33115-33122, 2005). Maximal ATPase activity was obtained with NH₄⁺ as activating cation. The enzyme was also stimulated by Na⁺, but in contrast to the rat enzyme, hardly by K⁺. SCH 28080 inhibited the NH₄⁺-stimulated activity of the human enzyme much more potently than that of the rat enzyme. The steady-state phosphorylation level of the human enzyme decreased with increasing pH, [K⁺], and [Na⁺] and nearly doubled in the presence of oligomycin. Oligomycin increased the sensitivity of the phosphorylated intermediate to ADP, demonstrating that it inhibited the conversion of E₁P to E₂P. All three cations stimulated the dephosphorylation rate dose-dependently. Our studies support a role of the human enzyme in H⁺/Na⁺ and/or H⁺/NH₄⁺ transport but not in Na⁺/K⁺ transport.     

Differences between CTP and ATP as substrates for the (Na + K)-ATPase

CTP was a poorer substrate than ATP when substituted in the (Na + K)-ATPase reaction assay, not only in terms of K_m but also of V. CDP was a poorer inhibitor than ADP, so product inhibition cannot account for CTP being a poorer substrate. In the Na-ATPase reaction, which the enzyme also catalyzes, substituting CTP for ATP resulted in greater activity, arguing against CTP being less effective than ATP in forming the enzyme-phosphate intermediate common to both reactions. Ligands that favor the E₂ conformational state of the enzyme, K⁺, Mg²⁺, and Mn₂⁺, inhibited the (Na + K)-CTPase reaction more than the (Na + K)-ATPase. Conversely, Triton X-100, which favors the E₁ conformational state of the enzyme, K⁺, Mg²⁺, and Mn²⁺, inhibited the (Na + K)-CTPase ATPase reaction but stimulated the (Na + K)-CTPase. Although the (Na + K)-ATPase reaction sequence probably involves cyclical interconversion between E₁ and E₂ conformational states (and is thus inhibitable by ligands favoring either state), the K-phosphatase reaction catalyzed by the enzyme apparently functions entirely in the E₂ state. This reaction is better stimulated by CTP plus Na⁺ than by ATP plus Na⁺; moreover, CTP lessens inhibition by Triton X-100, and ATP lessens inhibition by inorganic phosphate (which reacts with the E₂ state). These observations indicate that CTP is a poorer substrate than ATP because it is less effective in promoting conversion of E₂ to E₁, essential for the (Na + K)-dependent reaction mechanism. However, contrary to this rationale, dimethyl sulfoxide stimulated the (Na + K)-CTPase reaction although by other criteria, including inhibition of the (Na + K)-ATPase, the reagent appears to favor the E₂ over the E₁ conformational state.     

Regulation by the exogenous polyamine spermidine of Na,K-ATPase activity from the gills of the euryhaline swimming crab Callinectes danae (Brachyura, Portunidae)

Euryhaline crustaceans rarely hyporegulates and employ the driving force of the Na,K-ATPase, located at the basal surface of the gill epithelium, to maintain their hemolymph osmolality within a range compatible with cell function during hyper-regulation. Since polyamine levels increase during the adaptation of crustaceans to hyperosmotic media, we investigate the effect of exogenous polyamines on Na,K-ATPase activity in the posterior gills of Callinectes danae, a euryhaline swimming crab. Polyamine inhibition was dependent on cation concentration, charge and size in the following order: spermine > spermidine > putrescine. Spermidine affected K_0.5 values for Na⁺ with minor alterations in K_0.5 values for K⁺ and NH₄⁺, causing a decrease in maximal velocities under saturating Na⁺, K⁺ and NH₄⁺ concentrations. Phosphorylation measurements in the presence of 20 µM ATP revealed that the Na,K-ATPase possesses a high affinity site for this substrate. In the presence of 10 mM Na⁺, both spermidine and spermine inhibited formation of the phosphoenzyme; however, in the presence of 100 mM Na⁺, the addition of these polyamines allowed accumulation of the phosphoenzyme. The polyamines inhibited pumping activity, both by competing with Na⁺ at the Na⁺-binding site, and by inhibiting enzyme dephosphorylation. These findings suggest that polyamine-induced inhibition of Na,K-ATPase activity may be physiologically relevant during migration to fully marine environments.     

Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: I. Sodium Binding is Associated with a Conformational Rearrangement

To investigate Na⁺ binding to the ion-binding sites presented on the cytoplasmic side of the Na,K-ATPase, equilibrium Na⁺-titration experiments were performed using two fluorescent dyes, RH421 and FITC, to detect protein-specific actions. Fluorescence changes upon addition of Na⁺ in the presence of various Mg²⁺ concentrations were similar and could be fitted with a Hill function. The half-saturating concentrations and Hill coefficients determined were almost identical. As RH421 responds to binding of a Na⁺ ion to the third neutral site whereas FITC monitors conformational changes in the ATP-binding site or its environment, this result implies that electrogenic binding of the third Na⁺ ion is the trigger for a structural rearrangement of the ATP-binding moiety. This enables enzyme phosphorylation, which is accompanied by a fast occlusion of the Na⁺ ions and followed by the conformational transition E₁/E₂ of the protein. The coordinated action both at the ion and the nucleotide binding sites allows for the first time a detailed formulation of the mechanism of enzyme phosphorylation that occurs only when three Na⁺ ions are bound. Received: 8 October 1998/Revised: 29 December 1998     

Solubilization and further chromatographic purification of highly purified,membrane-bound Na,K-ATPase

Highly purified membrane-bound Na,K-ATPase from pig kidney outer medulla was dissolved in the non-ionic detergent C₁₂E₈. Chromatography of the dissolved material on a DEAE matrix yielded enzymatical material having a ouabain-binding capacity of 6.9 nmoles per mg protein (measured according to Lowry et al., with bovine serum albumin as standard). This material, which after addition of lipids had the same K⁺-phosphatase turnover as the membrane-bound enzyme, could consist entirely of live molecules with a molecular weight of 145 kDa, a value close to that expected for αβ-protomers of Na,K-ATPase.     

The E2P-like state induced by magnesium fluoride complexes in the Na,K-ATPase. Kinetics of formation and interaction with Rb+

The first X-ray crystal structures of the Na,K-ATPase were obtained in the presence of magnesium and fluoride as E2(K₂)Mg–MgF₄, an E2∙Pi-like state capable to occlude K⁺ (or Rb⁺). This work presents a functional characterization of the crystallized form of the enzyme and proposes a model to explain the interaction between magnesium, fluoride and Rb⁺ with the Na,K-ATPase. We studied the effect of magnesium and magnesium fluoride complexes on the E1–E2 conformational transition and the kinetics of Rb⁺ exchange between the medium and the E2(Rb₂)Mg–MgF₄ state. Our results show that both in the absence and in the presence of Rb⁺, simultaneous addition of magnesium and fluoride stabilizes the Na,K-ATPase in an E2 conformation, presumably the E2Mg–MgF₄ complex, that is unable to shift to E1 upon addition of Na⁺. The time course of conformational change suggests the action of fluoride and magnesium at different steps of the E2Mg–MgF₄ formation. Increasing concentrations of fluoride revert along a sigmoid curve the drop in the level of occluded Rb⁺ caused by Mg^2 +. Na⁺-induced release of Rb⁺ from E2(Rb₂)Mg–MgF₄ occurs at the same rate as from E2(Rb₂) but is insensitive to ADP. The rate of Rb⁺ occlusion into the E2Mg–MgF₄ state is 5–8 times lower than that described for the E2Mg–vanadate complex. Since the E2Mg–MgF₄ and E2Mg–vanadate complexes represent different intermediates in the E2-P → E2 dephosphorylation sequence, the variation in occlusion rate could provide a tool to discriminate between these intermediates.     

Estradiol modulates the sodium pump in the heart sarcolemma

Cardiovascular effects of estrogens and particularly that of estradiol involve protection of the heart against ischemia. These effects were believed to be mainly indirect, mediated via changes in the blood and blood vessels. In the present paper a direct action of estradiol on the heart is demonstrated. Estradiol stimulates (p < 0.001) the Na,K-ATPase activity of cardiac sarcolemmal membranes by stimulating in an allosteric manner, the activation of the enzyme by potassium. The latter activation involves also an increase in affinity to potassium of the potassium binding sites on the enzyme molecule, but remains without any effect on the capacity and K_Dvalue of specific ouabain binding to the Na,K-ATPase. Estradiol is also antagonizing the depression of Na,K-ATPase activity that may be caused by ischemia and it is stimulating (p < 0.01) the ouabain-sensitive uptake of ⁸⁶Rb into the heart cells.Our results indicate, that in addition to the known indirect effects of estradiol on the heart, the hormone also stimulates the activity and improves the kinetics of interaction of cardiac sarcolemmal Na,K-ATPase with ATP as well as with Na⁺ and K⁺ ions. This direct action may also account for the cardioprotective effects of estradiol.     

Effects of γ-irradiation on Na,K-ATPase in cardiac sarcolemma

Previous studies showed that adverse effect of ionizing radiation on the cardiovascular system is beside other factors mostly mediated by reactive oxygen and nitrogen species, which deplete antioxidant stores. One of the structures highly sensitive to radicals is the Na,K-ATPase the main system responsible for extrusion of superfluous Na⁺ out of the cell which utilizes the energy derived from ATP. The aim of present study was the investigation of functional properties of cardiac Na,K-ATPase in 20-week-old male rats 6 weeks after γ-irradiation by a dose 25 Gy (IR). Irradiation induced decrease of systolic blood pressure from 133 in controls to 85 mmHg in IR group together with hypertrophy of right ventricle (RV) and hypotrophy of left ventricle (LV). When activating the cardiac Na,K-ATPase with substrate, its activity was lower in IR in the whole concentration range of ATP. Evaluation of kinetic parameters revealed a decrease of the maximum velocity (V _max) by 40 % with no changes in the value of Michaelis–Menten constant (K _m). During activation with Na⁺, we observed a decrease of the enzyme activity in hearts from IR at all tested Na⁺ concentrations. The value of V _max decreased by 38 %, and the concentration of Na⁺ that gives half maximal reaction velocity (K _Na) increased by 62 %. This impairment in the affinity of the Na⁺-binding site together with decreased number of active Na,K-ATPase molecules, as indicated by lowered V _max values, are probably responsible for the deteriorated efflux of the excessive Na⁺ from the intracellular space in hearts of irradiated rats.     

Effects of PKA phosphorylation on the conformation of the Na,K-ATPase regulatory protein FXYD1

FXYD1 (phospholemman) is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the Na,K-ATPase enzyme complex in specific tissues and specific physiological states. In heart and skeletal muscle sarcolemma, FXYD1 is also the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinase A and by protein kinase C, which phosphorylate the protein at conserved Ser residues in its cytoplasmic domain, altering its Na,K-ATPase regulatory activity. FXYD1 adopts an L-shaped α-helical structure with the transmembrane helix loosely connected to a cytoplasmic amphipathic helix that rests on the membrane surface. In this paper we describe NMR experiments showing that neither PKA phosphorylation at Ser68 nor the physiologically relevant phosphorylation mimicking mutation Ser68Asp induces major changes in the protein conformation. The results, viewed in light of a model of FXYD1 associated with the Na,K-ATPase α and β subunits, indicate that the effects of phosphorylation on the Na,K-ATPase regulatory activity of FXYD1 could be due primarily to changes in electrostatic potential near the membrane surface and near the Na⁺/K⁺ ion binding site of the Na,K-ATPase α subunit.     

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.     

Effects of Free Radicals on Partial Reactions of the Na,K-ATPase

The function of the Na,K-ATPase is known to be considerably impaired in the presence of free radicals such as OH^•. While previous experiments were largely based on the loss of enzymatic activity of the protein, this is the first communication dealing with partial reactions of the pump cycle in the presence of free radicals produced by water radiolysis. Three different system states, which are directly involved in ion transfer catalyzed by the enzyme, showed similar sensitivity to free radical action. This is indicated by largely identical D₃₇-doses of the decay of the reaction amplitudes investigated. The decrease in the efficiency of the enzyme functions was largely due to a lethal damage of pump molecules. A kinetic analysis of the ATP-induced conformational transition E₁→ E₂ revealed, however, that a minor component of the inactivation is due to a reduction of the transition rate constant. The decrease of the enzymatic activity could be simulated by the decay of the rate-limiting conformational transition. This finding indicates the conservation of a close coupling between ATP-hydrolysis and sodium translocation process throughout free-radical induced inactivation. As a result of the tight coupling, enzyme modification at different system states leads to similar functional consequences for the protein. Received: 19 July 1996/Revised: 21 October 1996     

Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements

Summary Membrane fragments containing a high density of Na, K-ATPase can be noncovalently labeled with amphiphilic styryl dyes (e.g., RH 421). Phosphorylation of the Na,K-ATPase by ATP in the presence of Na⁺ and in the absence of K⁺ leads to a large increase of the fluorescence of RH 421 (up to 100%). In this paper evidence is presented that the styryl dye mainly responds to changes of the electric field strength in the membrane, resulting from charge movements during the pumping cycle: (i) The spectral characteristic of the ATP-induced dye response essentially agrees with the predictions for an electrochromic shift of the absorption peak. (ii) Adsorption of lipophilic anions to Na, K-ATPase membranes leads to an increase, adsorption of lipophilic cations to the decrease of dye fluorescence. These ions are known to bind to the hydrophobic interior of the membrane and to change the electric field strength in the boundary layer close to the interface. (iii) The fluorescence change that is normally observed upon phosphorylation by ATP is abolished at high concentrations of lipophilic ions. Lipophilic ions are thought to redistribute between the adsorption sites and water and to neutralize in this way the change of field strength caused by ion translocation in the pump protein. (iv) Changes of the fluorescence of RH 421 correlate with known electrogenic transitions in the pumping cycle, whereas transitions that are known to be electrically silent do not lead to fluorescence changes. The information obtained from experiments with amphiphilic styryl dyes is complementary to the results of electrophysiological investigations in which pump currents are measured as a function of transmembrane voltage. In particular, electrochromic dyes can be used for studying electrogenic processes in microsomal membrane preparations which are not amenable to electrophysiological techniques.Deceased (September 13, 1990).     

Tryptic digestion of the (Na + K)-ATPase is both sensitive to and modifies K+ interactions with the enzyme

Tryptic digestion of the (Na + K)-ATPase in the presence of choline chloride or NaCl (Na-type) and in the presence of KCl (K-type) produced distinct patterns of peptide fragments and losses of catalytic activity. TheK _0.5 for K⁺ to shift digestion from the Na-type, and its sensitivity to dimethyl sulfoxide and Triton X-100, were consistent with K⁺ acting at sites on the cytoplasmic face of the enzyme through which the K-phosphatase reaction also is activated. Reagents favoring the E₁ conformational states, oligomycin, Triton, and ATP, shifted the pattern toward the Na-type, whereas those favoring E₂ states, dimethyl sulfoxide, MgCl₂, and MnCl₂, shifted the pattern toward the K-type. Na-type digestion caused a greater loss of K-phosphatase than (Na + K)-ATPase activity, and the residual K-phosphatase activity was more sensitive to inhibition by Triton and ATP but stimulated more by dimethyl sulfoxide and inhibited less by P_i and MnCl₂; all these effects are consistent with such digestion shifting equilibria toward E₁ enzyme states. Accordingly, theK _0.5 for K⁺ to activate the (Na + K)-ATPase was increased. However, theK _0.5 for the K-phosphatase was unchanged; this observation requires revision of previous formulations, and bears on additional aspects of enzyme activity as well.     

Effects of Ganoderma Lucidum shell-broken spore on oxidative stress of the rabbit urinary bladder using an in vivo model of ischemia/reperfusion

The present study was oriented to gender specificity of Na,K-ATPase in cerebellum, the crucial enzyme maintaining the intracellular homeostasis of Na ions in healthy and diabetic Wistar rats. The effects of diabetes on properties of the Na,K-ATPase in cerebellum derived from normal and streptozotocin (STZ)-diabetic rats of both genders were investigated. The samples were excised at different time intervals of diabetes induced by STZ (65 mg kg^?1) for 8 days and 16 weeks. In acute 8-day-lasting model of diabetes, Western blot analysis showed significant depression of α1 isoform of Na,K-ATPase in males only. On the other hand, concerning the activity, the enzyme seems to be resistant to the acute model of diabetes in both genders. Prolongation of diabetes to 16 weeks was followed by increasing the number of active molecules of Na,K-ATPase exclusively in females as indicated by enzyme kinetic studies. Gender specificity was observed also in nondiabetic animals revealing higher Na,K-ATPase activity in control males probably caused by higher number of active enzyme molecules as indicated by increased value of V _max when comparing to control female group. This difference seems to be age dependent: at the age of 16 weeks, the V _max value in females was higher by more than 90%, whereas at the age of 24 weeks, this difference amounted to only 28%. These data indicate that the properties of Na,K-ATPase in cerebellum, playing crucial role in maintaining the Na⁺ and K⁺ gradients, depend on gender, age, and duration of diabetic impact.     

E2P State Stabilization by the N-terminal Tail of the H,K-ATPase ��-Subunit Is Critical for Efficient Proton Pumping under in Vivo Conditions

The catalytic α-subunits of Na,K- and H,K-ATPase require an accessory β-subunit for proper folding, maturation, and plasma membrane delivery but also for cation transport. To investigate the functional significance of the β-N terminus of the gastric H,K-ATPase in vivo, several N-terminally truncated β-variants were expressed in Xenopus oocytes, together with the S806C α-subunit variant. Upon labeling with the reporter fluorophore tetramethylrho da mine-6-maleimide, this construct can be used to determine the voltage-dependent distribution between E₁P/E₂P states. Whereas the E₁P/E₂P conformational equilibrium was unaffected for the shorter N-terminal deletions βΔ4 and βΔ8, we observed significant shifts toward E₁P for the two larger deletions βΔ13 and βΔ29. Moreover, the reduced ΔF/F ratios of βΔ13 and βΔ29 indicated an increased reverse reaction via E₂P → E₁P + ADP → E₁ + ATP, because cell surface expression was completely unaffected. This interpretation is supported by the reduced sensitivity of the mutants toward the E₂P-specific inhibitor {"type":"entrez-protein","attrs":{"text":"SCH28080","term_id":"1053015931","term_text":"SCH28080"}}SCH28080, which becomes especially apparent at high concentrations (100 μm). Despite unaltered apparent Rb⁺ affinities, the maximal Rb⁺ uptake of these mutants was also significantly lowered. Considering the two putative interaction sites between the β-N terminus and α-subunit revealed by the recent cryo-EM structure, the N-terminal tail of the H,K-ATPase β-subunit may stabilize the pump in the E₂P conformation, thereby increasing the efficiency of proton release against the million-fold proton gradient of the stomach lumen. Finally, we demonstrate that a similar truncation of the β-N terminus of the closely related Na,K-ATPase does not affect the E₁P/E₂P distribution or pump activity, indicating that the E₂P-stabilizing effect by the β-N terminus is apparently a unique property of the H,K-ATPase.The gastric H,K-ATPase fulfills the remarkable task of pumping protons against a more than 10⁶-fold concentration gradient. H⁺ extrusion is coupled to countertransport of an equal number of K⁺ ions for each ATP molecule hydrolyzed, resulting in an electroneutral overall process (1). Characteristic for all P-type ATPases, the enzyme cycles between the two principal conformational states (E₁ and E₂) and the corresponding phosphointermediates (E₁P and E₂P), which are formed by reversible phosphorylation of an aspartate residue in the highly conserved DKTGTLT motif. According to a Post-Albers-like reaction scheme (see Fig. 1A), the conformational E₁P → E₂P transition converts the high H⁺/low K⁺ affinity of the cation binding pocket into a low H⁺/high K⁺ affinity binding site, hence enabling proton release into the stomach lumen and subsequent binding of extracellular K⁺. Because the pump faces a lumenal proton concentration of ∼150 mm (2), proton release is probably the energetically most demanding step in the reaction cycle. Thus, during the conformational E₁P → E₂P transition, enormous pK_a changes of the H⁺-coordinating residues have to occur that most likely involve the rearrangement of a positively charged lysine side chain (Lys-791 in rat H,K-ATPase) (3).Open in a separate windowFIGURE 1.Post-Albers scheme (A) and cryo-EM structural representation of pig gastric H,K-ATPase in the fluoroaluminate-bound pseudo-E₂P state (B). A, Post-Albers scheme of the proposed reaction cycle of the gastric H,K-ATPase. E₁P/E₂P conformational states giving rise to voltage jump-induced fluorescence changes of TMRM-labeled H,K-ATPase molecules are highlighted (gray box). B, structural representation based on the cryo-EM structure of the pig gastric H,K-ATPase (surface or mesh, contoured at 1 σ; EM Data Bank code 5104) and the corresponding homology model (schematic; Protein Data Bank code 3IXZ). Inset, a close-up view (from the right side of the molecule) showing the putative interaction sites of the β-subunit N terminus with the P-domain (red arrow) and αTM3 (black arrow), respectively. Color coding is indicated in the figure.All P₂-type ATPases share a common catalytic α-subunit, composed of 10 transmembrane domains harboring the ion-binding sites and a large cytoplasmic loop with the nucleotide-binding domain, the phosphorylation domain (P-domain),² and the actuator domain (A-domain) (4). However, a unique feature of K⁺-transporting Na,K- and H,K-ATPase enzymes is the requirement for an accessory β-subunit, which is indispensable for proper folding, maturation, and plasma membrane delivery (5, 6). Despite only 20–30% overall sequence identity between the H,K-ATPase β-subunit and the Na,K β-isoforms, the topogenic structure is similar: a short N-terminal cytoplasmic tail, followed by a single transmembrane segment and a large extracellular C-terminal domain with glycosylation sites and disulfide-bridging cysteines. Numerous studies have demonstrated that the β-subunit of the Na,K-ATPase is more than just a chaperone for the α-subunit, being also required for proper ion transport activity of the holoenzyme. In fact, it has been discovered that different cell- and tissue-specific β-isoforms have distinct effects on the cation affinities (7–9). Furthermore, it was shown that mutational changes in all three topogenic domains of the Na,K-ATPase β-subunit (10–19) as well as chemical interference with disulfide-forming cysteines in the Na,K-ATPase β-subunit ectodomain (20–22) affect the cation transport properties of the sodium pump. Finally, conformational changes in the β-subunit during the Na,K-ATPase reaction cycle were demonstrated by proteolytic digestion studies (23) and voltage clamp fluorometry (24).Far less is known about the functional significance of the single H,K-ATPase β-isoform, especially about its potential impact on cation transport (reviewed in Refs. 25 and 26). We have proven recently that E₂P state-specific transmembrane interactions between residues in αTM7 and two highly conserved tyrosines in the βTM of both Na,K- and H,K-ATPase significantly stabilize the E₂P conformation (19). Mutational disruptions of this interaction resulted in substantial shifts toward E₁P and severely affected H⁺ secretion, which highlighted the physiological relevance of this E₂P state stabilization. Notably, according to the recently published cryo-EM structure of pig gastric H,K-ATPase in the pseudo-E₂P state (27), the N-terminal tail of the β-subunit makes direct contact with the phosphorylation domain of the α-subunit (see Fig. 1B), thus indicating an additional E₂P state stabilization mediated by the β-N terminus. Although this idea was further supported by biochemical studies on N-terminally truncated mutants, direct evidence for this putative E₂P-stabilizing interaction and its potential significance for ion transport in intact cells is still lacking.Here, we demonstrate for the first time the functional importance of the gastric H,K-ATPase β-subunit N terminus in living cells under in vivo conditions: voltage clamp fluorometry, Rb⁺ flux, and {"type":"entrez-protein","attrs":{"text":"SCH28080","term_id":"1053015931","term_text":"SCH28080"}}SCH28080 sensitivity measurements revealed E₁P-shifted, ion transport-impaired phenotypes for two N-terminally truncated H,K β-variants, thus substantiating the E₂P-stabilizing effect of the β-N terminus suggested by the recent cryo-EM structure.     

E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2

This work presents a detailed kinetic study that shows the coupling between the E2→E1 transition and Rb⁺ deocclusion stimulated by Na⁺ in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb⁺ and the increase in eosin fluorescence (the formation of E1) as a function of time. The E2→E1 transition and Rb⁺ deocclusion are described by the sum of two exponential functions with equal amplitudes, whose rate coefficients decreased with increasing [Rb⁺]. The rate coefficient values of the E2→E1 transition were very similar to those of Rb⁺-deocclusion, indicating that both processes are simultaneous. Our results suggest that, when ATP is absent, the mechanism of Na⁺-stimulated Rb⁺ deocclusion would require the release of at least one Rb⁺ ion through the extracellular access prior to the E2→E1 transition. Using vanadate to stabilize E2, we measured occluded Rb⁺ in equilibrium conditions. Results show that, while Mg^2 + decreases the affinity for Rb⁺, addition of vanadate offsets this effect, increasing the affinity for Rb⁺. In transient experiments, we investigated the exchange of Rb⁺ between the E2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the E2→E1 transition caused by Na⁺ without significantly affecting the rate of Rb⁺ deocclusion. On the other hand, we found the first evidence of a very low rate of Rb⁺ occlusion in the enzyme–vanadate complex, suggesting that this complex would require a change to an open conformation in order to bind and occlude Rb⁺.     

On the lack of inotropy of cardiac glycosides on skeletal muscle: a comparison of Na+, K+-ATPases from skeletal and cardiac muscle.

Comparison of Na,K-ATPase from skeletal and cardiac muscle revealed that, although the skeletal muscle enzyme was only slightly less sensitive to inhibition by ouabain, the rates of [³H]ouabain binding to, and dissociation from, the skeletal enzyme were much faster than the corresponding rates for the cardiac enzyme. The skeletal muscle enzyme required higher concentrations of potassium to stabilize the ouabainenzyme complex and to stimulate the K⁺-phosphatase activity. The K⁺-phosphatase activity was only 8% of the Na,K-ATPase activity of the skeletal muscle enzyme, compared to 22% for the cardiac preparation. The glycoprotein subunit found in Na,K-ATPases from cardiac and many other tissues appeared to be absent in the enzyme from skeletal muscle. The differences in binding and dissociation rates for ouabain suggest that there may be significant differences in the structure of the digitalis receptor in the two enzymes. The I₅₀ for ouabain inhibition of the skeletal muscle Na,K-ATPase was, however, only slightly higher than for the cardiac enzyme, suggesting that the lack of an inotropic effect of cardiac glycosides on skeletal muscle could not be due to failure of the digitalis drugs to bind to and inhibit the membrane-linked sodium pump.     

Na,K-ATPase structure/function relationships probed by the denaturant urea

Urea interacts with the Na,K-ATPase, leading to reversible as well as irreversible inhibition of the hydrolytic activity. The enzyme purified from shark rectal glands is more sensitive to urea than Na,K-ATPase purified from pig kidney. An immediate and reversible inhibition under steady-state conditions of hydrolytic activity at 37 °C is demonstrated for the three reactions studied: the overall Na,K-ATPase activity, the Na-ATPase activity observed in the absence of K⁺ as well as the K⁺-dependent phosphatase reaction (K-pNPPase) seen in the absence of Na⁺. Half-maximal inhibition is seen with about 1 M urea for shark enzyme and about 2 M urea for pig enzyme. In the presence of substrates there is also an irreversible inhibition in addition to the reversible process, and we show that ATP protects against the irreversible inhibition for both the Na,K-ATPase and Na-ATPase reaction, whereas the substrate paranitrophenylphosphate leads to a slight increase in the rate of irreversible inhibition of the K-pNPPase. The rate of the irreversible inactivation in the absence of substrates is much more rapid for shark enzyme than for pig enzyme. The larger number of potentially urea-sensitive hydrogen bonds in shark enzyme compared to pig enzyme suggests that interference with the extensive hydrogen bonding network might account for the higher urea sensitivity of shark enzyme. The reversible inactivation is interpreted in terms of domain interactions and domain accessibilities using as templates the available crystal structures of Na,K-ATPase. It is suggested that a few interdomain hydrogen bonds are those mainly affected by urea during reversible inactivation.