ADP- and K+-sensitive phosphorylated intermediate of Na,K-ATPase

In the phosphoenzyme (EP) of the electric eel Na,K-ATPase, the sum of the ADP-sensitive EP and the K+-sensitive EP exceeds 150% of EP in the presence of 100 mM Na+. This unusual phenomenon can be explained by the formation of three phosphoenzymes: ADP-sensitive K+-insensitive (E1P), K+-sensitive ADP-insensitive (E2P), and ADP- and K+-sensitive (E*P) phosphoenzymes, as proposed by N?rby et al. (N?rby, J. G., Klodos, I., and Christiansen, N. O. (1983) J. Gen. Physiol. 82, 725-757). By applying a simple approximation method for the assay of E1P, E*P, and E2P, it was found that the phosphorylation of the enzyme was much faster than the conversion among each EP and the phosphoenzyme changed as E1NaATP----E1P----E*P----E2P. In the fragmental eel enzyme, the step of E*P to E2P was much slower than the step of E1P to E*P. In the steady state, the E1P was predominant above 400 mM Na+, whereas E*P and E2P were predominant between 60 and 300 mM Na+ and below 60 mM Na+, respectively. The characteristic difference of the eel enzyme from the beef brain enzyme and probably from the kidney enzyme seems to be that the dissociation constant of Na+ on the E1P-E*P equilibrium is higher than that on the E*P-E2P. The E*P and E1P both interacted with ADP to form ATP without formation of inorganic phosphate in the absence of free Mg2+. In the Na,K-ATPase proteoliposomes, the vesicle membrane interfered with the conversion of E1P to E2P, especially the change of E1P to E*P, and furthermore, the E1P content increased. This barrier effect was partially counteracted by monensin or carbonyl cyanide m-chlorophenylhydrazone. Oligomycin reacted with E1P and probably with E*P, therefore inhibiting their conversion to E2P and interaction with K+.     

Two different phosphorylation-dephosphorylation cycles of Na,K-ATPase proteoliposomes accompanying Na+ transport in the absence of K+

The phosphorylated intermediate (EP) of the Na,K-ATPase proteoliposomes (PL) prepared from the electric eel enzyme is composed of an ADP-sensitive K+-insensitive form (E1P), an ADP- and K+-sensitive form (E*P), and a K+-sensitive ADP-insensitive form (E2P). The composition of the intermediate varied with the cholesterol content of the lipid bilayer. The PL containing less than 30 mol % cholesterol (LCPL) formed E2P-rich EP in the presence of 10 mM Na+ on both sides at 15 degrees C, while the PL containing more than 35 mol % cholesterol (HCPL) formed E*P-rich EP under the same condition. In the presence of ionophore (monensin, nigericin, A23187), the HCPL formed E2P-rich EP as reported in the preceding paper. The turnover rate of Na-ATPase activity (the ratio of Na-ATPase to the EP level) in the LCPL was lower than that in the HCPL, and the addition of 20 microM monensin or A23187 to the HCPL reduced the Na-ATPase activity. The coupling ratio of Na+ influx (cellular efflux):Na+ efflux (cellular influx):ATP hydrolysis was 2.8:1.8:1 in the LCPL, although 1.6:0.6:1 in the HCPL. The coupling ratio of Na+ influx:ATP hydrolysis in the HCPL increased to 2.8:1 in the presence of A23187. Moreover, the increase of ATP concentration enhanced not only the Na-ATPase activity in the LCPL and HCPL with monensin but also the Na+ influx in the LCPL. This ATP enhancement was not found, however, in the HCPL without ionophores. The ADP enhancement of the Na+ influx was not observed in either the HCPL or the LCPL. We conclude from these observations that there are at least two different phosphorylation-dephosphorylation cycles (an E2P cycle and an E*P cycle) in the PL in the absence of K+. The E2P cycle transports three Na+ from the extravesicular (cytoplasmic) to the intravesicular (extracellular) side and two Na+ in the opposite direction per cycle and is similar to the ATP-dependent Na+-Na+ exchange system already reported (Blostein, R. (1983) J. Biol. Chem. 258, 7948-7953; Cornelius, F., and Skou, J. C. (1985) Biochim. Biophys. Acta 818, 211-221). However, the E*P cycle transports one Na+ from the extravesicular to the intravesicular side/cycle and has not yet been previously reported.     

Cytoplasmic K+ effects on phosphoenzyme of Na,K-ATPase proteoliposomes and on the Na+-pump activity

Three phosphorylated reaction intermediates (EP) of Na,K-ATPase, and ADP-sensitive K+-insensitive EP (E1P), an ADP- and K+-sensitive EP (E*P), and a K+-sensitive ADP-insensitive EP (E2P), have been discovered at present. By using Na,K-ATPase proteoliposomes (PL) prepared from the electric eel enzyme, we found in this study that E*P existed even in the presence of K+ on both sides of the PL and that there was a sidedness difference in K+ sites between E*P and E2P. Cytoplasmic K+ (K+cyt) accelerated the conversion of E*P to E2P but did not dephosphorylate the E2P. Although the extracellular K+ accelerated the dephosphorylation of E2P, it did not interact with E*P directly. This K+cyt effect was also verified by the activation of Na+-pump in the Na+-K+ exchange mode. In the presence of K+cyt, both the ATP hydrolysis and Na+ uptake rates of the PL containing K+ inside vesicles increased sigmoidally with the concentrations of ATP and cytoplasmic Na+ (Na+cyt). However, in the absence of K+cyt, these Na+-pump reactions in PL containing K+ inside vesicles had only a hyperbolic curve. These results imply that the E*P to E2P conversion is one of the rate-limiting steps of the Na+-pump in the presence of a high concentration of ATP and that K+cyt may control this reaction step by enhancing the conversion rate of E*P to E2P.     

Na+ and K+ transport at basolateral membranes of epithelial cells. II. K+ efflux and stoichiometry of the Na,K-ATPase

Changes of 42K efflux (J23K) caused by ouabain and/or furosemide were measured in isolated epithelia of frog skin. From the kinetics of 42K influx (J32K) studied first over 8-9 h, K+ appeared to be distributed into readily and poorly exchangeable cellular pools of K+. The readily exchangeable pool of K+ was increased by amiloride and decreased by ouabain and/or K+-free extracellular Ringer solution. 42K efflux studies were carried out with tissues shortcircuited in chambers. Ouabain caused an immediate (less than 1 min) increase of the 42K efflux to approximately 174% of control in tissues incubated either in SO4-Ringer solution or in Cl-Ringer solution containing furosemide. Whereas furosemide had no effect on J23K in control tissues bathed in Cl-rich or Cl-free solutions, ouabain induced a furosemide-inhibitable and time-dependent increase of a neutral Cl-dependent component of the J23K. Electroconductive K+ transport occurred via a single-filing K+ channel with an n' of 2.9 K+ efflux before ouabain, normalized to post-ouabain (+/- furosemide) values of short-circuit current, averaged 8-10 microA/cm2. In agreement with the conclusions of the preceding article, the macroscopic stoichiometry of ouabain-inhibitable Na+/K+ exchange by the pump was variable, ranging between 1.7 and 7.2. With increasing rates of transepithelial Na+ transport, pump-mediated K+ influx saturated, whereas Na+ efflux continued to increase with increases of pump current. In the usual range of transepithelial Na+ transport, regulation of Na+ transport occurs via changes of pump-mediated Na+ efflux, with no obligatory coupling to pump-mediated K+ influx.     

Na+ and K+ transport at basolateral membranes of epithelial cells. I. Stoichiometry of the Na,K-ATPase

The stoichiometry of pump-mediated Na/K exchange was studied in isolated epithelial sheets of frog skin. 42K influx across basolateral membranes was measured with tissues in a steady state and incubated in either beakers or in chambers. The short-circuit current provided estimates of Na+ influx at the apical membranes of the cells. 42K influx of tissues bathed in Cl- or SO4-Ringer solution averaged approximately 8 microA/cm2. Ouabain inhibited 94% of the 42K influx. Furosemide was without effect on pre-ouabain-treated tissues but inhibited a ouabain-induced and Cl--dependent component of 42K influx. After taking into account the contribution of the Na+ load to the pump by way of basolateral membrane recycling of Na+, the stoichiometry was found to increase from approximately 2 to 6 as the pump-mediated Na+ transport rate increased from 10 to 70 microA/cm2. Extrapolation of the data to low rates of Na+ transport (less than 10 microA/cm2) indicated that the stoichiometry would be in the vicinity of 3:2. As pump-mediated K+ influx saturates with increasing rates of Na+ transport, Na+ efflux cannot be obligatorily coupled to K+ influx at all rates of transepithelial Na+ transport. These results are similar to those of Mullins and Brinley (1969. Journal of General Physiology. 53:504-740) in studies of the squid axon.     

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.     

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).     

TNP-8N3-ADP photoaffinity labeling of two Na,K-ATPase sequences under separate Na+ plus K+ control

ATP has high- and low-affinity effects on the sodium pump and other P-type ATPases. We have approached this question by using 2',3'-O-(trinitrophenyl)-8-azidoadenosine 5'-diphosphate (TNP-8N(3)-ADP) to photoinactivate and label Na,K-ATPase, both in its native state and after covalent FITC block of its high-affinity ATP site. With the native enzyme, the photoinactivation rate constant increases hyperbolically with a K(D(TNP-8N)3(-)(ADP)) of 0.11 microM; TNP-ATP and ATP protect the site with high affinities. The inactivation does not require Na(+), but K(+) inhibits with a K(K)' of 12 microM; Na(+) reverses this effect, with a K(Na) of 0.17 mM. This pattern suggests that Na(+) and K(+) are binding at sites in their "intracellular" conformation. It was known that FITC did not abolish the reverse phosphorylation by P(i), or the K(+)-phosphatase activity, and that TNP-8N(3)-ADP could subsequently photoinactivate the latter with >100-fold lower affinity; in that case, the cation sites acted as if facing outward [Ward, D. G., and Cavieres, J. D. (1998) J. Biol. Chem. 273, 14277-14284, 33759-33765]. Native and FITC-modified enzymes have now been photolabeled with TNP-8N(3)-[alpha-(32)P]ADP and alpha-chain soluble tryptic peptides separated by reverse-phase HPLC. With native Na,K-ATPase, three labeled peaks lead to the unique sequence alpha-(470)Ile-Val-Glu-Ile-Pro-Phe-Asn-Ser-Thr-Asn-X-Tyr-Gln-Leu-Ser-Ile-His-Lys(487), the dropped residue being alphaLys480. With the FITC enzyme, instead, two independent labeling and purification cycles return the sequence alpha-(721)Ala-Asp-Ile-Gly-Val-Ala-Met-Gly-Ile-Ala-Gly-Ser-Asp-Val-Ser-Lys(736). These results suggest that Na,K-ATPase also has a low-affinity nucleotide binding region, one that is under distinctive allosteric control by Na(+) and K(+). Moreover, the cation effects seem compatible with a slow, passive Na(+)/K(+) carrier behavior of the FITC-modified sodium pump.     

Na,K-ATPase and the development of Na+ transport in rat distal colon

Summary Na, K-ATPase function was studied in order to evaluate the mechanism of increased colonic Na⁺ transport during early postnatal development. The maximum Na⁺-pumping activity that was represented by the equivalent short-circuit current after addition of nystatin (I _sc ^N ) did not change during postnatal life or after adrenalectomy performed in 16-day-old rats.I _sc ^N was entirely inhibited by ouabain; the inhibitory constant was 0.1mm in 10-day-old (young) and 0.4mm in 90-day-old (adult) rats. The affinity of the Na, K pump for Na⁺ was higher in young (11mm) than in adult animals (19mm). The Na, K-ATPase activity (measured after unmasking of latent activity by treatment with sodium dodecylsulfate) increased during development and was also not influenced by adrenalectomy of 16-day-old rats. The inhibitory constant for ouabain (K _I ) was not changed during development (0.1–0.3mm). Specific [³H]ouabain binding to isolated colonocytes increased during development (19 and 82 pmol/mg protein), the dissociation constant (K _D ) was 8 and 21 m in young and adult rats, respectively. The Na⁺ turnover rate per single Na, K pump, which was calculated fromI _sc ^N and estimated density of binding sites per cm² of tissue was 500 in adult and 6400 Na⁺/min·site in young rats. These data indicate that the very high Na⁺ transport during early postnatal life reflects an elevated turnover rate and increased affinity for Na⁺ of a single isoform of the Na, K pump. The development of Na⁺ extrusion across the basolateral membrane is not directly regulated by corticosteroids.     

Nonenzymatic glycation of Na,K-ATPase. Effects on ATP hydrolysis and K+ occlusion

Glycation of the Na,K-ATPase in vitro (formation of Schiff base with glucose followed by reduction with NaCNBH3) shows the presence of three classes of reactive amino groups that differentially affect catalysis and cation binding. Reaction in the absence of ATP results in irreversible inhibition of enzyme activity with a t1/2 of 53 min. This is due to modification of one class of amino groups that affect the catalytic domain of the enzyme. In the presence of ATP, glycation first results in a shift in the steady state kinetics of ATP hydrolysis from substrate activation to Michaelis-Menten kinetics accompanied by an increase in the apparent affinity for K+ in the p-nitrophenylphosphatase reaction. This change in kinetic properties occurs with a t1/2 of 9 min and results in the complete loss of K+ occlusion. Incorporation of glucose is into the catalytic subunit, remote from the N-terminal end. Apparent total inhibition of K+ occlusion occurs with a stoichiometry 0.8 mol of glucose incorporated per mol of enzyme. Therefore, there is a rapidly reacting amino group that affects the cation binding domain of the Na,K-ATPase. More slowly, with a t1/2 of 9 h, the ATP hydrolysis kinetics change from Michaelis-Menten to substrate inhibition without recovery of K+ occlusion, showing that, in the E1 conformation, there is a third, slower reacting class of amino groups in the Na,K-ATPase that affects low affinity nucleotide interaction with the catalytic subunit.     

Uncoupled Na+-efflux on reconstituted shark Na,K-ATPase is electrogenic

In liposomes with reconstituted shark Na,K-ATPase produced to contain sucrose addition of external Na+ and ATP induce an uncoupled Na+-efflux on inside-out oriented pumps which can be inhibited by digitoxigenin. This flux mode is found to be electrogenic and accompanied by hydrolysis of ATP. The coupling ratio of Nacyt transported per ATP split is 3:1 measured as the initial rate of rise in transmembrane potential and initial rate of liberated Pi.     

Thyroid hormone regulates alpha and alpha + isoforms of Na,K-ATPase during development in neonatal rat brain

The brain contains two molecular forms of Na,K-ATPase designated alpha found in non-neuronal cells and neuronal soma and alpha + found in axolemma. Previously we have shown that the abundance of both forms (determined by immunoblots) as well as Na,K-ATPase activity increases 10-fold between 4 days before and 20 days after birth (Schmitt, C. A., and McDonough, A. A. (1986) J. Biol. Chem. 261, 10439-10444). Hypothyroidism in neonates blunts these increases. Neonatal, but not adult brain Na,K-ATPase is thyroid hormone (triiodothyronine, T3) responsive. This study defines the period during which brain Na,K-ATPase responds to T3. The start of the critical period was defined by comparing Na,K-ATPase activity and alpha and alpha + abundance in hypothyroid and euthyroid neonates (birth to 30 days of age). For all parameters, euthyroid was significantly higher by 15 days of age. The end of the critical period was defined by dosing hypothyroid neonates with T3 daily (0.1 micrograms/g body weight) beginning at increasing days of age, and sacrificing all at 30 days then assaying enzyme activity and abundance. Those starting T3 treatment on or before day 19 were restored to euthyroid levels of Na,K-ATPase activity and abundance, while those starting T3 treatment on or after day 22 remained at hypothyroid levels of enzyme activity and abundance. We conclude that brain Na,K-ATPase alpha and alpha + isoforms are sensitive to T3 by as late as 15 days of age and that the period of thyroid hormone responsiveness is over by 22 days.     

Protective effect of a low K+ cardioplegic solution on myocardial Na,K-ATPase activity.

Long duration ischemia in hypothermic conditions followed by reperfusion alters membrane transport function and in particular Na,K-ATPase. We compared the protective effect of two well-described cardioplegic solutions on cardiac Na,K-ATPase activity during reperfusion after hypothermic ischemia. Isolated perfused rat hearts (n = 10) were arrested with CRMBM or UW cardioplegic solutions and submitted to 12 hr of ischemia at 4 degrees C in the same solution followed by 60 min of reperfusion. Functional recovery and Na,K-ATPase activity were measured at the end of reperfusion and compared with control hearts and hearts submitted to severe ischemia (30 min at 37 degrees C) followed by reflow. Na,K-ATPase activity was not altered after 12 hr of ischemia and 1 hr reflow when the CRMBM solution was used for preservation (55 +/- 2 micromolPi/mg prot/hr) compared to control (53 +/- 2 micromol Pi/mg prot/hr) while it was significantly altered with UW solution (44 +/- 2 micromol Pi/mg prot/hr, p < 0.05 vs control and CRMBM). Better preservation of Na,K-ATPase activity with the CRMBM solution was associated with higher functional recovery compared to UW as represented by the recovery of RPP, 52 +/- 12% vs 8 +/- 5%, p < 0.05 and coronary flow (70 +/- 2% vs 50 +/- 8%, p < 0.05). The enhanced protection provided by CRMBM compared to UW may be related to its lower K+ content.     

Metabolic depression and Na+/K+ gradients in the aestivating Australian goldfields frog,Neobatrachus wilsmorei

During aestivation the metabolic rate of the Australian goldfields frog Neobatrachus wilsmorei was reduced by 80% from its standard metabolic rate. The in vitro rate of oxygen consumption of isolated muscle and skin from aestivating frogs was up to 50% lower than that of the non-aestivating frogs. This in vitro rate of oxygen consumption was maintained for 6–12 h, indicating an intrinsic metabolic depression of tissues during aestivation. Frogs became dehydrated during aestivation. Muscle, skin and liver also became dehydrated during aestivation, but brain and kidney did not. Na⁺ and K⁺ contents and extracellular space measurement for muscle indicated that ion gradients were maintained across the muscle cell membrane during aestivation. Increases in plasma concentrations of Na⁺ and K⁺ were matched with similar increases in muscle intracellular ion concentrations. Extracellular space measurements were unsuccessful in the other tissues, but K⁺ content in all tissues (per dry weight) was maintained during aestivation, and the concentration of plasma K⁺ did not increase above that which can be accounted for by dehydration, indicating that K⁺ gradients were maintained.Abbreviations bm body mass - DPM disintegrations per minute - dw dry weight - MR metabolic rate - vO₂ rate of oxygen consumption - ww wet weight     

Perturbation of Na+ and K+ gradients in human fibroblasts incubated in unsupplemented saline solutions

Changes in the intracellular concentrations of Na+ and K+ of fetal human fibroblasts have been followed after replacement of serum-containing growth media with unsupplemented and serum-supplemented saline solution (Earle's balanced salt solution). Incubation in unsupplemented salt solution was followed by a progressive increase of the internal Na+ counterbalanced by a decrease of internal K+, without major alterations of the internal osmolarity. After 3 h incubation the intracellular Na+ and K+ concentrations were 120 mM and 50 mM, respectively. These intracellular ion derangements were not associated with a failure of the (Na+ + K+)-ATPase pump, whose activity actually increased with enhanced intracellular Na+ concentration. Ion changes did not take place when serum (in excess of 0.5%, final concentration) was present in the saline solution and a complete restoration to normal of the Na+ and K+ gradients occurred upon addition of serum to cells previously incubated in plain saline solution. The effects of serum were mimicked by furosemide, thus suggesting that channels sensitive to this diuretic are involved in the movement of Na+ and K+ following fibroblast incubation in unsupplemented saline solution.     

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.     

Short-term NO synthase inhibition and the Na+-binding properties of cardiac Na,K-ATPase

It is known that hypertension is accompanied by increased [Na+]i. The functional properties of Na,K-ATPase, which transports the Na+ out and K+ into myocardial cells during the relaxation phase, were investigated in the left ventricle (LV), septum (SV) and the right ventricle (RV) of anesthetized dogs with moderate acute blood pressure elevation elicited by short-term (4-hour) NO synthase inhibition. The NO-insufficiency was induced by administration of an L-arginine analogue, the N(G)-nitro-L-arginine methyl ester (L-NAME). Concerning the function of Na,K-ATPase under the conditions of lowered NO synthesis, we focused our attention to the binding of Na+ to the enzyme molecule. Activation of the enzyme by increasing Na+ concentrations revealed significant changes in both the maximal velocity (Vmax) and the affinity for Na+ (K(Na)) in all investigated heart sections. The Vmax increased by 27% in LV, by 87% in SV and by 58% in RV. The K(Na) value increased by 86% in LV, by 105% in SV and by 93% in RV, indicating an apparent decrease in the sensitivity of the Na+-binding site in the Na,K-ATPase molecule. This apparently decreased pump affinity for Na+ together with the increase of Vmax suggest that, during the short-term inhibition of NO synthesis, the Na,K-ATPase is capable of extruding the excessive Na+ from the myocardial cells more effectively at higher [Na+]i, as compared to the Na,K-ATPase of control animals.