Time-dependent increases in Na+,K(+)-ATPase content of low-frequency-stimulated rabbit muscle.

Chronic low-frequency stimulation of rabbit fast-twitch muscle induced time-dependent increases in the concentration of the sarcolemmal Na+,K(+)-ATPase and in mitochondrial citrate synthase activity. The almost twofold increase in Na+,K(+)-ATPase preceded the rise in citrate synthase and was complete after 10 days of stimulation. We suggest that the increase in Na+,K(+)-ATPase enhances resistance to fatigue of low-frequency-stimulated muscle prior to elevations in aerobic-oxidative capacity.     

Ultrastructural localization of Na+,K+-ATPase in rat and rabbit kidney medulla

Na+,K+-ATPase was localized at the ultrastructural level in rat and rabbit kidney medulla. The cytochemical method for the K+-dependent phosphatase component of the enzyme, using p-nitrophenylphosphate (NPP) as substrate, was employed to demonstrate the distribution of Na+, K+- ATPase in tissue-chopped sections from kidneys perfusion-fixed with 1% paraformaldehyde-0.25% glutaraldehyde. In other outer medulla of rat kidney, ascending thick limbs (MATL) were sites of intense K+-dependent NPPase (K+-NPPase) activity, whereas descending thick limbs and collecting tubules were barely reactive. Although descending thin limbs (DTL) of short loop nephrons were unstained, DTL from long loop nephrons in outer medulla were sites of moderate K+-NPPase activity. In rat inner medulla, DTL and ascending thin limbs (ATL) were unreactive for K+-NPPase. In rabbit medulla, only MATL were sites of significant K+-NPPase activity. The specificity of the cytochemical localization of Na+,K+-ATPase at reactive sites in rat and rabbit kidney medulla was demonstrated by K+-dependence of reaction product deposition, localization of reaction product (precipitated phosphate hydrolyzed from NPP) to the cytoplasmic side of basolateral plasma membranes, insensitivity of the reaction to inhibitors of nonspecific alkaline phosphatase, and, in the glycoside-sensitive rabbit kidney, substantial inhibition of staining by ouabain. The observed pattern of distribution of the sodium transport enzyme in kidney medulla is particularly relevant to current models for urine concentration. The presence of substantial Na+,K+-ATPase in MATL is consistent with the putative role of this segment as the driving force for the countercurrent multiplication system in the outer medulla. The absence of significant activity in inner medullary ATL and DTL, however, implies that interstitial solute accumulation in this region probably occurs by passive processes. The localization of significant Na+,K+-ATPase in outer medullary DTL of long loop nephrons in the rat suggests that solute addition in this segment may occur in part by an active salt secretory mechanism that could ultimately contribute to the generation of inner medullary interstitial hypertonicity and urine concentration.     

Na+,K(+)-ATPase activity in human colorectal carcinoma

Na+,K(+)-ATPase activities in macroscopically unchanged mucosa (conditionally normal tissue) and human colorectal carcinoma (mainly low-grade and moderately differentiated adenocarcinomas) have been investigated. Microsomal fractions are similar by dimensions of the membrane fragments detected by photon correlation spectroscopy analysis. The activation optima under digitonin pretreatment of the membrane fractions differ significantly for Na+,K(+)-ATPase and concomitant Mg(2+)-ATPase activity, but are the same in conditionally normal and cancerous tissues. This allows to detect correctly total levels of the Na+,K(+)-ATPase activity in the detergent-pretreated preparations. The moderate decrease of the Na+,K(+)-ATPase activity is revealed in carcinomas. It is concluded that a decrease of activity of the ouabain-sensitive human Na+,K(+)-ATPase is characteristic of colorectal carcinoma.     

Kidney Na+,K(+)-ATPase is associated with moesin

Na+,K(+)-ATPase is a ubiquitous plasmalemmal membrane protein essential for generation and maintenance of transmembrane Na+ and K+ gradients in virtually all animal cell types. Activity and polarized distribution of renal Na+,(+)-ATPase appears to depend on connection of ankyrin to the spectrin-based membrane cytoskeleton as well as on association with actin filaments. In a previous study we showed copurification and codistribution of renal Na+,K(+)-ATPase not only with ankyrin, spectrin and actin, but also with two further peripheral membrane proteins, pasin 1 and pasin 2. In this paper we show by sequence analysis through mass spectrometry as well as by immunoblotting that pasin 2 is identical to moesin, a member of the FERM (protein 4.1, ezrin, radixin, moesin) protein family, all members of which have been shown to serve as cytoskeletal adaptor molecules. Moreover, we show that recombinant full-length moesin as well as its FERM domain bind to Na+,K(+)-ATPase and that this binding can be inhibited by an antibody specific for the ATPase activity-containing cytoplasmic loop (domain 3) of the Na+,K(+)-ATPase alpha-subunit. This loop has been previously shown to be a site essential for ankyrin binding. These observations indicate that moesin might not only serve as direct linker molecule of Na+,K(+)-ATPase to actin filaments but also modify ankyrin binding at domain 3 of Na+,K(+)-ATPase in a way similar to protein 4.1 modifying the binding of ankyrin to the cytoplasmic domain of the erythrocyte anion exchanger (AE1).     

Temperature dependence of the rates of conformational changes reported by fluorescein 5'-isothiocyanate modification of H+,K(+)- and Na+,K(+)-ATPases.

Stopped-flow fluorometry has been used to measure the forward and reverse rates of the conformational change from E1 to E2 in the fluorescein-modified proton and sodium pumps (1) as a function of Na+ and K+ concentrations to verify the proposed mechanism of ion interaction with the enzymes and (2) as a function of temperature to gain insight into the nature of the conformational transition. (1) The fluorescence changes caused by Na+ and K+ are consistent with rapid competitive binding of the two ions to the E1 conformations of the enzymes followed by rate-limiting transitions between E1K and E2K. (2) Reaction coordinate diagrams for the E1K to E2K transitions in the H,K-ATPase and Na,K-ATPase are qualitatively similar. Enthalpy barriers to reaction are partially compensated by increased entropy in the transition states. However, there are striking quantitative differences between the two enzymes. The E2K to E1K reaction of the H,K-ATPase is more than 2 orders of magnitude faster (tau 1/2 = 6 ms at 22 degrees C) than the reverse rate of the Na,K-ATPase transition (tau 1/2 = 1.6 s), explaining repeated failure to detect a K(+)-"occluded" form of the H,K-enzyme. The E2K conformer of the Na,K-ATPase is 3 orders of magnitude more stable than E1K, while the E1K and E2K conformations of the H,K-ATPase are nearly equivalent energetically.     

Kinetics of K(+) occlusion by the phosphoenzyme of the Na(+),K(+)-ATPase

Investigations of K⁺-occlusion by the phosphoenzyme of Na⁺,K⁺-ATPase from shark rectal gland and pig kidney by stopped-flow fluorimetry reveal major differences in the kinetics of the two enzymes. In the case of the pig enzyme, a single K⁺-occlusion step could be resolved with a rate constant of 342 (±26) s⁻¹. However, in the case of the shark enzyme, two consecutive K⁺-occlusions were detected with rate constants of 391 (±19) s⁻¹ and 48 (±2) s⁻¹ at 24°C and pH 7.4. A conformational change of the phosphoenzyme associated with K⁺-occlusion is, thus, the major rate-determining step of the shark enzyme under saturating concentrations of all substrates, whereas for the pig enzyme the major rate-determining step under the same conditions is the E2 → E1 transition and its associated K⁺ deocclusion and release to the cytoplasm. The differences in rate constants of the K⁺ occlusion reactions of the two enzymes are paralleled by compensating changes to the rate constant for the E2 → E1 transition, which explains why the differences in the enzymes' kinetic behaviors have not previously been identified.     

The inhibitory monoclonal antibody M7-PB-E9 stabilizes E2 conformational states of Na+,K(+)-ATPase.

Monoclonal antibody M7-PB-E9 binds the sheep kidney Na+,K(+)-ATPase alpha-subunit with high affinity (Kd = 3 nM) and inhibits enzyme turnover in competition with ATP, and, like ATP, in the presence of Mg2+, it stimulates the rate of ouabain binding [Ball, W. J. (1984) Biochemistry 23, 2275-2281]. In this study, covalent attachment of fluorescein 5'-isothiocyanate (FITC) at (or near) the enzyme's ATP binding site did not alter the antibody's affinity for alpha nor did bound antibody alter the anisotropy of (r = 0.36) or the solvent accessibility of iodide to bound FITC. Further, in its E1Na+ conformation (4 mM NaCl), the enzyme's affinity for the ATP congener eosin was unaltered by the bound antibody (Kd = 9 nM). In contrast, partial E2 conformations induced by KCl lowered eosin affinities (0.2 mM KCl, Kd = 28 nM; 0.4 mM, Kd = 86 nM), and M7-PB-E9 reduced these affinities further (Kd = 66 and 130 nM, respectively). By monitoring the fluorescence changes of the FITC-labeled enzyme, the antibody was found to assist several ligand-induced conformational transitions from E1 (E1Na+ or E1Tris) to E2 (E2K+, E2-P(i)Mg2+, or E2Mg2+.ouabain) states, and inhibit the E2K(+)-->E1Na+ transition. Antibody binding alone, however, did not appear to significantly alter enzyme conformation. The antibody therefore is not directed against the ATP site but binds to a region of alpha distinct from any ligand binding site and which plays an important role in the E1<-->E2 transitions.     

Digitalis sensitivity of Na+,K(+)-ATPase, myocytes and the heart.

Cardiac Na+,K(+)-ATPase, the receptor molecule for digitalis glycosides, have isoforms with different intrinsic affinities for the glycosides. Expression of these isoforms are under developmental and hormonal regulation. Switching in isoforms to those with lower intrinsic affinity may decrease digitalis sensitivity of the heart. In addition to the intrinsic affinity of the cardiac Na+,K(+)-ATPase for the glycoside, increases in the rate of Na+ influx and decreases in extracellular K+ concentrations increase glycoside sensitivity of the heart and also reduces the margin of safety by reducing reserve capacity of the sodium pump. Reserve capacity of the sodium pump is also reduced by pathological conditions or aging, resulting in reduced margin of safety for the glycoside. Events that follow sodium pump inhibition also affect sensitivity of the heart to digitalis toxicity. These are hypercalcemia and magnesium depletion. It is now feasible to predict digitalis sensitivity of the heart, not empirically but based on the understanding of the mechanisms responsible for the positive inotropic and toxic actions of the glycoside.     

Interaction of sodium and potassium ions with Na+,K(+)-ATPase. IV. Affinity change for K+ and Na+ of Na+,K(+)-ATPase in the cycle of the ATP hydrolysis reaction

The Kd for ouabain-sensitive K+ or Rb+ binding to Na+,K(+)-ATPase was determined by the centrifugation method with radioactive K+ and Rb+ in the presence of various combinations of Na+, ATP, adenylylimidodiphosphate (AMPPNP), adenylyl-(beta,gamma-methylene)diphosphonate (AMPPCP), Pi, and Mg2+. From the results of the K+ binding experiments, Kd for Na+ was estimated by using an equation describing the competitive inhibition between the K+ and Na+ binding. 1) The Kd for K+ binding was 1.9 microM when no ligand was present. Addition of 2 mM Mg2+ increased the Kd to 15-17 microM. In the presence of 2 mM Mg2+, addition of 3 mM AMPPCP with or without 3 mM Na+ increased the Kd to 1,000 or 26 microM, respectively. These Kds correspond to those for K+ of Na.E1.AMPPCPMg or E1.AMPPCPMg, respectively. 2) Addition of 4 mM ATP with or without 3 mM Na+ decreased the Kd from 15-17 microM to 5 or 0.8 microM, respectively. Because the phosphorylated intermediate was observed but ATPase activity was scarcely observed in the K+ binding medium containing 3 mM ATP and 2 mM Mg2+ in the absence of Na+ as well as in the presence of Na+ at 0 degrees C, it is suggested that K+ binds to E2-P.Mg under these ligand conditions. 3) The Kd for Na+ of the enzyme in the presence of 3 mM AMPPCP or 4 mM ATP with Mg2+ was estimated to be 80 or 570 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na+, K+-ATPase: relation of conformational transitions to function

Summary In its native environment, Na⁺, K⁺-ATPase of the plasma membrane is an oligomer consisting of two or more of each of two major subunits. Na⁺ and K⁺ move across the membrane through the channels that exist between the catalytic subunits of this oligomer. Two distinct ligand-induced conformational transitions (one due to the binding of K⁺ and ATP to the enzyme, and the other resulting from the phosphorylation of the enzyme in the presence of Na⁺ and ATP) cause changes in the geometries of the intersubunit channels, and provide the necessary energy-linked gating mechanisms for the transmembrane movements of ions against electrochemical gradients.     

Kinetics of oligomycin inhibition and activation of Na+/K(+)-ATPase.

Oligomycin inhibition of the maximal hydrolysis activity of ox brain Na+/K(+)-ATPase was studied at varying NaCl concentrations and it was found that for a given amount of live enzyme, the observed inhibition of a particular total oligomycin concentration decreased as the amount of added, (heat-) denatured enzyme increased. In the present article we derive a scale factor for the oligomycin concentration, i.e., the fraction of the total concentration of oligomycin which is free in solution, as a function of the enzyme concentration used. This fraction decreased linearly with the protein concentration and may attain quite small values. We also study the Na(+)-dependence of the hydrolysis rate at saturating substrate concentrations ([Mg2+] = [ATP] = 3 mM), in the presence as well as the absence of KCl, at various concentrations of oligomycin. These data may be explained if it is assumed that the sole effect of oligomycin is to confer upon the enzyme an increased affinity for Na+, i.e., oligomycin merely enhances the inhibitory effect of Na+ on the (maximal) activity seen at high Na(+)-concentrations. The increased Na(+)-affinity in the presence of oligomycin should result in activation of the hydrolysis rate measured under conditions where Na(+)-activation is predominant, i.e., at low Na(+)-concentration and sub-saturating substrate concentrations. This prediction is verified for both Na(+)-ATPase and for Na+/K(+)-ATPase. This proposed action of oligomycin seems to be corroborated also by other evidence discussed in the text.     

Hydrolysis by acylphosphatase of erythrocyte membrane Na+, K(+)-ATPase phosphorylated intermediate.

Acylphosphatase, purified from human erythrocytes, actively hydrolyzes the phosphoenzyme intermediate of human red blood cell membrane Na+, K(+)-ATPase. This effect occurred with acylphosphatase amounts (up to 10 units/mg membrane protein) that fall within the physiological range. Acylphosphatase addition to erythrocyte membranes resulted in a significant increase in the rate of Na+, K(+)-dependent ATP hydrolysis. Maximal stimulation, observed with 10 units/mg membrane protein, was of about 80% over basal value. The same acylphosphatase amount enhanced of about 40% the rate of ATP driven Na+ transport into inside out red cell membrane vesicles. Taken together these findings suggest a potential role of acylphosphatase in the control of the activity of erythrocyte membrane Na,K pump.     

A marine algal Na(+)-activated ATPase possesses an immunologically identical epitope to Na+,K(+)-ATPase.

Immunological homology was investigated between Heterosigma akashiwo (a marine algae) Na(+)-activated ATPase and animal Na+,K(+)-ATPase. The former polypeptide [(1989) Plant Cell Physiol. 30, 923-928] reacted with anti-serum raised against the amino-terminal half of the pig kidney Na+,K(+)-ATPase alpha subunit. It is suggested that the Na+,K(+)-ATPase epitope within the amino-terminal region is conserved in the plant Na(+)-activated ATPase, and the region containing the epitope may be important for Na ion transport.     

Isolation and characterization of a cDNA encoding the putative distal colon H+,K(+)-ATPase. Similarity of deduced amino acid sequence to gastric H+,K(+)-ATPase and Na+,K(+)-ATPase and mRNA expression in distal colon, kidney, and uterus.

A series of Northern blot hybridization experiments using probes derived from the rat gastric H+,K(+)-ATPase cDNA and the human ATP1AL1 gene revealed the presence of a 4.3-kilobase mRNA in colon that seemed likely to encode the distal colon H+,K(+)-ATPase, the enzyme responsible for K+ absorption in mammalian colon. A rat colon library was then screened using a probe from the ATP1AL1 gene, and cDNAs containing the entire coding sequence of a new P-type ATPase were isolated and characterized. The deduced polypeptide is 1036 amino acids in length and has an Mr of 114,842. The protein exhibits 63% amino acid identity to the gastric H+,K(+)-ATPase alpha-subunit and 63% identity to the three Na+,K(+)-ATPase alpha-subunit isoforms, consistent with the possibility that it is a K(+)-transporting ATPase. Northern blot analyses show that the 4.3-kilobase mRNA is expressed at high levels in distal colon; at much lower levels in proximal colon, kidney, and uterus; and at trace levels in heart and forestomach. The high mRNA levels in distal colon and the similarity of the colon pump to both gastric H+,K(+)- and Na+,K(+)-ATPases suggest that it is the distal colon H+,K(+)-ATPase. Furthermore, expression of its mRNA in kidney raises the possibility that the enzyme also corresponds to the H+,K(+)-ATPase that seems to play a role in K+ absorption and H+ secretion in the distal nephron.     

Mutational effects on conformational changes of the dephospho- and phospho-forms of the Na+,K+-ATPase

Gly263 of the rat kidney Na(+),K(+)-ATPase is highly conserved within the family of P-type ATPases. Mutants in which Gly263 or the juxtaposed Arg264 had been replaced by alanine were expressed at high levels in COS-1 cells and characterized functionally. Titrations of Na(+),K(+), ATP, and vanadate dependencies of Na(+),K(+)-ATPase activity showed changes in the apparent affinities relative to wild-type compatible with a displacement of the E(1)-E(2) conformational equilibrium in favor of E(1). The level of the K(+)-occluded form was reduced in the Gly263-->Ala and Arg264-->Ala mutants, and the rate constant characterizing deocclusion of K(+) or Rb(+) was increased as much as 20-fold in the Gly263-->Ala mutant. Studies of the sensitivity of the phosphoenzyme to K(+) and ADP showed a displacement of the E(1)P-E(2)P equilibrium of the phosphoenzyme in favor of E(1)P, and dephosphorylation experiments carried out at 25 degrees C on a millisecond time scale using a quenched-flow technique demonstrated a reduction of the E(1)P to E(2)P conversion rate in the mutants. Hence, the mutations displaced the conformational equilibria of dephosphoenzyme and phosphoenzyme in parallel in favor of the E(1) and E(1)P forms. The observed effects were more pronounced in the Gly263-->Ala mutant compared with the Arg264-->Ala mutant. Leu332 mutations that likewise displaced the conformational equilibria in favor of E(1) and E(1)P were also studied. Unlike the Gly263-->Ala mutant the Leu332 mutants displayed a wild-type like rate of K(+) deocclusion. Thus, the effect of the Gly263 mutation on the E(1)-E(2) conformational equilibrium seems to be caused mainly by an acceleration of the K(+)-deoccluding step, whereas in the Leu332 mutants the rate of the reverse reaction seems to be reduced.     

Pig kidney Na+,K+-ATPase. Primary structure and spatial organization

cDNAs complementary to pig kidney mRNAs coding for alpha- and beta-subunits of Na+,K+-ATPase were cloned and sequenced. Selective tryptic hydrolysis of the alpha-subunit within the membrane-bound enzyme and tryptic hydrolysis of the immobilized isolated beta-subunit were also performed. The mature alpha- and beta-subunits contain 1016 and 302 amino acid residues, respectively. Structural data on the peptides from extramembrane regions of the alpha-subunit and on glycopeptides of the beta-subunit underlie a model for the transmembrane arrangement of Na+,K+-ATPase polypeptide chains.     

Allosteric effect of ATP on Na(+),K(+)-ATPase conformational kinetics

The kinetics of the E2 --> E1 conformational change of unphosphorylated Na+,K+-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 degrees C). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize the E2 conformation. When rabbit enzyme was mixed with 130 mM NaCl alone or with 130 mM NaCl and varying concentrations of Na2ATP simultaneously, a fluorescence decrease was observed. In the absence of ATP, the fluorescence decrease followed a biexponential time course, but at ATP concentrations after mixing of >or=50 microM, the fluorescence transient could be adequately fitted by a single exponential. On the basis of the agreement between theoretical simulations and experimental traces, we propose that in the absence of bound ATP the conformational transition occurs as a two step reversible process within a protein dimer, E2:E2 --> E2:E1 --> E1:E1. In the presence of 130 mM NaCl, the sum of the forward and backward rate constants for the E2:E2 --> E2:E1 and E2:E1 --> E1:E1 transitions were found to be 10.4 (+/-1.0) and 0.49 (+/-0.02) s-1, respectively. At saturating concentrations of ATP, however, the transition occurs in a single reversible step with the sum of its forward and backward rate constants equal to 35.2 (+/-0.3) s-1. It was found that ATP acting at a high affinity site (Kd approximately 0.25 microM), stimulated the reverse reaction, E1ATP --> E2ATP, in addition to its known allosteric low affinity (Kd approximately 71 microM) stimulation of the forward reaction, E2ATP --> E1ATP.