Extracellular Allosteric Na Binding to the Na,K-ATPase in Cardiac Myocytes

Whole-cell patch-clamp measurements of the current, I_p, produced by the Na⁺,K⁺-ATPase across the plasma membrane of rabbit cardiac myocytes show an increase in I_p over the extracellular Na⁺ concentration range 0–50 mM. This is not predicted by the classical Albers-Post scheme of the Na⁺,K⁺-ATPase mechanism, where extracellular Na⁺ should act as a competitive inhibitor of extracellular K⁺ binding, which is necessary for the stimulation of enzyme dephosphorylation and the pumping of K⁺ ions into the cytoplasm. The increase in I_p is consistent with Na⁺ binding to an extracellular allosteric site, independent of the ion transport sites, and an increase in turnover via an acceleration of the rate-determining release of K⁺ to the cytoplasm, E2(K⁺)₂ → E1 + 2K⁺. At normal physiological concentrations of extracellular Na⁺ of 140 mM, it is to be expected that binding of Na⁺ to the allosteric site would be nearly saturated. Its purpose would seem to be simply to optimize the enzyme’s ion pumping rate under its normal physiological conditions. Based on published crystal structures, a possible location of the allosteric site is within a cleft between the α- and β-subunits of the enzyme.     

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.     

Molecular Mechanisms of the Redox Regulation of the Na,K-ATPase

Biophysics - This review considers the molecular mechanisms involved in the redox regulation of the Na,K-ATPase. The enzyme creates a transmembrane gradient of sodium and potassium ions, which is...     

Glucose regulation of pre-steady state kinetics of ATP hydrolysis by Na,K-ATPase

The effect of glucose and 2-deoxy-D-glucose on pre-steady state kinetics of ATP hydrolysis by Na,K-ATPase has been investigated by following pH transients in a stopped-flow spectrophotometer. A typical pre-steady state signal showed an initial decrease then subsequent increase in acidity. Under optimal Na^＋ （120 mM） and K^＋ （30 mM） concentrations, magnitudes of both H^＋ release and H^＋ absorption were found to be approximately 1.0/ATPase molecule. The presence of 1 mM glucose significantly decreased H^＋ absorption at high Na^＋ concentrations, whereas it was ineffective at low Na^＋. H^＋ release was decreased significantly in the presence of 1 mM glucose at Na^＋ concentrations ranging from 30 mM to 120 mM. Similar to the control, K^＋ did not show any effect on either H^＋ release or H^＋ absorption at all tested combinations of Na^＋ and K^＋ concentrations. Pre-steady state H^＋ signal obtained in the presence of 2-deoxy-D-glucose did not vary significantly as compared with glucose. Delayed addition of K^＋ （by 30 ms） to the mixture （enzyme＋ 120 mM Na^＋ATP＋glucose） showed that only small fractions of population absorb H^＋ in the absence of K^＋. No H^＋ absorption was observed in the absence of Na^＋. Delayed mixing of Na^＋ or K^＋ did not have any effect on H^＋ release. Effect of 2-deoxy-D-glucose on H^ absorption and release was almost the same as that of glucose at all combinations of Na^＋ and K^＋ concentrations. Results obtained have been discussed in terms of an extended kinetic scheme which shows that, in the presence of either glucose or 2-deoxy-D-glucose, significantly fewer enzyme molecules reache the E-P（3Na＋） stage and that K^ plays an important role in the conversion of E1 .ADP.P（3Na^＋） to H^＋.E1-（3Na^＋） complex.     

N-acetylimidazole inactivates renal Na,K-ATPase by disrupting ATP binding to the catalytic site

Treatment of renal Na,K-ATPase with N-acetylimidazole (NAI) results in loss of Na,K-ATPase activity. The inactivation kinetics can be described by a model in which two classes of sites are acetylated by NAI. The class I sites are rapidly reacting, the acetylation is prevented by the presence of ATP (K0.5 congruent to 8 microM), and the inactivation is reversed by incubation with hydroxylamine. These data suggest that the class I sites are tyrosine residues at the ATP binding site. The second class of sites are more slowly reacting, not protected by ATP, nor reversed by hydroxylamine treatment. These are probably lysine residues elsewhere in the protein. The associated K-stimulated p-nitrophenylphosphatase activity is inactivated by acetylation of the class II sites only; thus the tyrosine residues associated with ATP binding to the catalytic center are not essential for phosphatase activity. Inactivated enzyme no longer has high-affinity ATP binding associated with the catalytic site, although low-affinity ATP effects (inhibition of phosphatase and deocclusion of Rb) are still present. The inactivated enzyme can still be phosphorylated by Pi, occlude Rb+ ions, and undergo the major conformational transitions between the E1 Na and E2 K forms of the enzyme. Thus acetylation of the Na,K-ATPase by NAI inhibits high-affinity ATP binding to the catalytic center and produces inactivation.     

Short-term NO synthase inhibition and the ATP affinity of cardiac Na,K-ATPase

It was previously shown that 4 hours lasting inhibition of nitric oxide synthesis by administration of an L-arginine analogue, the A(G)-nitro-L-arginine methyl ester (L-NAME) changed the affinity of the Na-binding site of Na,K-ATPase thus resulting in elevation of enzyme activity especially at higher concentrations of sodium. Using the same experimental model, we focused our attention in the present study to the question of binding of ATP to the enzyme molecule in the left ventricle (LV), ventricular septum (S) and the right ventricle (RV) of the dog heart. Activation of the enzyme by increasing concentrations of ATP revealed a significant increase of the Vmax only in septum (by 38 %). The K(M) increased significantly in septum (by 40 %) and in left ventricle (by 56 %) indicating an altered sensitivity of the ATP-binding site of Na,K-ATPase in the hearts of NO-deficient animals. The alterations of Na,K-ATPase in its ability to bind and hydrolyze ATP are localized to the tissue surrounding the cavity of the left ventricle.     

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+.     

18 O-exchange during ATP and n-nitrophenylphosphate hydrolysis by Na, K-ATPase from bovine brain]

The reaction of the oxygen isotope exchange (18O-exchange) was studied in the course of the Na, K-ATPase reaction. It was shown that the intermediary and direct 18O-exchanges occurred in the system in the presence of both ATP and p-NPP. These findings are indicative of the same intermediate during the hydrolytic process in both cases. The intermediary 18O-exchange was activated by N-ethylmaleimide, hydroxylamine and 2.0--1.5 18O atoms, respectively. The detection of 18O-exchange Ouabain had no effect on the exchange. The levels of intermediary 18O-exchange during ATP and p-NPP hydrolyses were equal to 1.3--1.4 and 2.0--1.5 18O atoms, respectively. The detection of 18O-exchange reactions at the intermediary steps of both ATP and p-NPP hydrolyses implies the identity of certain stages in the destruction of these substrates by Na, K-ATPase.     

Membrane compartmentalized ATP and its preferential use by the Na, K-ATPase of human red cell ghosts

This paper describes work which begins to define the molecular organization in the region of the membrane that comprises the functional domain of the Na:K pump. The membrane-bound phosphoglycerate kinase (PGK) and Na, K-ATPase appear to be directly linked via a compartmentalized form of ATP. Evidence for the membrane pool of ATP is based on the labeling characteristics of the phosphoproteins by [γ-(32)P]ATP of ghosts incubated under various conditions. Preincubation of ghosts in the presence of ATP at 37 degrees C, but not at 0 degrees C, completely obscures the formation of the Na-phosphoprotein in ghosts washed and subsequently incubated in the presence of [gamma-(32)P]ATP. In contrast to the Na component, the Mg component of phosphorylation is only slightly altered by preincubation with ATP. ATPase activity measured as (32)P(i) liberated during the subsequent incubation at 0 degrees C, reflects completely the differential effects of preincubation with ATP on (32)P incorporation into phosphoprotein. ATP placed within the pool by preincubation can be removed by operating the Na, K-ATPase or the PGK reaction in the reverse direction by use of exogenous substrates. Alternatively, the membrane pool of ATP can be formed also from exogenous substrates by running the PGK reaction in the forward direction. These results, while providing direct support for a membrane compartment of ATP, also indicate the location of this compartment in relation to the PGK and the Na, K-ATPase. In addition, these results also imply that the Mg and Na components are different enzymatic entities since substrate ATP can be derived from separate sources.     

Phosphorylation of the Na,K-ATPase and the H,K-ATPase

Phosphorylation is a widely used, reversible means of regulating enzymatic activity. Among the important phosphorylation targets are the Na⁺,K⁺- and H⁺,K⁺-ATPases that pump ions against their chemical gradients to uphold ionic concentration differences over the plasma membrane. The two pumps are very homologous, and at least one of the phosphorylation sites is conserved, namely a cAMP activated protein kinase (PKA) site, which is important for regulating pumping activity, either by changing the cellular distribution of the ATPases or by directly altering the kinetic properties as supported by electrophysiological results presented here. We further review the other proposed pump phosphorylations.     

Active transport of Na+ by modified Na,K-ATPase

The ability of ATP, CTP, ITP, GTP and UTP to induce ouabain-sensitive accumulation of Na+ by proteoliposomes with a reconstituted Na/K-pump was studied. At low Na+/K+ ratio (20 mM/50 mM), a correlation was observed between the proton-accepting capacity of the nucleotide and its efficiency as an active transport substrate. In order to test the hypothesis on the role of the negative charge in position 1 of the purine (3-pyrimidine) base of the nucleotide in the reversible transitions from the Na- to the K-conformations of Na,K-ATPase, two ATP analogs (N1-hydroxy-ATP possessing a proton-accepting ability and N1-methoxy-ATP whose molecule carries a negative charge quenched by a methyl group) were used. The first substrate provides for active accumulation of Na+ by proteoliposomes at a rate similar to that of ATP, whereas the second substrate is fairly ineffective.     

The Na,K-ATPase

The energy dependent exchange of cytoplasmic Na⁺ for extracellular K⁺ in mammalian cells is due to a membrane bound enzyme system, the Na,K-ATPase. The exchange sustains a gradient for Na⁺ into and for K⁺ out of the cell, and this is used as an energy source for creation of the membrane potential, for its de- and repolarisation, for regulation of cytoplasmic ionic composition and for transepithelial transport. The Na,K-ATPase consists of two membrane spanning polypeptides, an -subunit of 112-kD and a -subunit, which is a glycoprotein of 35-kD. The catalytic properties are associated with the -subunit, which has the binding domain for ATP and the cations. In the review, attention will be given to the biochemical characterization of the reaction mechanism underlying the coupling between hydrolysis of the substate ATP and transport of Na⁺ and K⁺.     

Stabilization of Na,K-ATPase by ionic interactions

The effect of ions on the thermostability and unfolding of Na,K-ATPase from shark salt gland was studied and compared with that of Na,K-ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 degrees C, as does the kidney enzyme at 42 degrees C (but not at 20 degrees C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both enzymes against this rapid inactivation. As detected by DSC, the shark enzyme undergoes thermal unfolding at lower temperature (Tm approximately 45 degrees C) than does the kidney enzyme (Tm approximately 55 degrees C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the kidney enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 degrees C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K-ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.     

Stabilization of Na,K-ATPase by ionic interactions

The effect of ions on the thermostability and unfolding of Na,K-ATPase from shark salt gland was studied and compared with that of Na,K-ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 °C, as does the kidney enzyme at 42 °C (but not at 20 °C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both enzymes against this rapid inactivation. As detected by DSC, the shark enzyme undergoes thermal unfolding at lower temperature (T_m ≈ 45 °C) than does the kidney enzyme (T_m ≈ 55 °C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the kidney enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 °C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K-ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.     

Temperature-dependence of ATP level, organic phosphate production and Na,K-ATPase in human lymphocytes

It was previously shown that human lymphocytes maintain a normal accumulation of K+ and exclusion of Na+ between 37 degrees and 10 degrees C., and a significant net accumulation of K+ and exclusion of Na+ at even lower temperatures. The studies reported here show that the level of ATP is near-normal for at least 24 hours between 37 degrees and 10 degrees C., but that ATP synthesis and utilization are progressively and markedly decreased with decreasing temperatures below 37 degrees C. The activities of the membrane Na+- and K+-activated ATPases have typical marked temperature-dependences. Therefore, the normal accumulation of K+ and exclusion of Na+ between 37 degrees and 10 degrees C., and the normal rate of Na+ efflux at these temperatures, do not correlate with properties of the Na+,K+-ATPase or with rates of synthesis and utilization of ATP.     

Study of ATP binding in the active site of Na,K-ATPase as probed by ultraviolet resonance Raman spectroscopy

The ultraviolet resonance Raman (UV RR) spectra of functional ATP/membrane-bound Na⁺K⁺-ATPase complexes have been obtained. The substrate binding in the enzyme active site has been shown to be accompanied with significant changes in the electronic vibrational structure of the adenine ring. From the spectral analysis of ATP, 8-Br-ATP and 6-NHMe-adenine at various pH values the conclusion was made that N1 and the NH₂, group and, probably, N7 of the substrate adenine part, interact with the protein surroundings via hydrogen bonds.     

Occlusion of Na+ by the Na,K-ATPase in the presence of oligomycin

Oligomycin occludes Na+ in an E1-form of the Na,K-ATPase. The rate constants for the release of Na+ from the E1-form and for the transition to the E2-form are about 0.5 s-1. The effect of oligomycin is not seen using other cations which also have a Na+-like effect on the enzyme conformation. The inhibitory effect of oligomycin on the ADP-ATP dependent Na:Na exchange but not on the accompanying ADP-ATP exchange can be explained from a decrease in the rate of release of Na+ from an E1 approximately phosphoform with Na+ occluded, E'1 approximately P (Na3), i.e. with Na+ in the membrane phase, to an E"1 approximately PNa3 form with Na+ not occluded. E"1 approximately PNa3 is at a step before formation of E2-P, and disappears at a high rate when ADP reacts with E"1 approximately P (Na3).     

The regulation of Na, K-ATPase activity by the substrate

The mechanism of functioning of Na, K-ATPase system is considered, the peculiarities of hydrolysis in different substrates are described. The experimental results testify to the role of substrate structure in E2----E1-transition, Na+ transport, K(+)-dependent phosphatase activity and quaternary structure of enzyme. The regulatory role of molecular organization of Na, K-ATPase in ion transport is discussed.     

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.