A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps

Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na(+)-dependent and proton-dependent secondary transport systems that are responsible for uptake and extrusion of metabolites and other ions. The ion gradients are also both directly and indirectly used to control pH homeostasis and to regulate cell volume. The plasma membrane H(+)-ATPase maintains a proton gradient in plants and fungi and the Na(+),K(+)-ATPase maintains a Na(+) and K(+) gradient in animal cells. Structural information provides insight into the function of these two distinct but related P-type pumps.     

Papain fragmentation of the (Na+,K+)-ATPase beta subunit reveals multiple membrane-bound domains

Purified dog kidney (Na+,K+)-ATPase was reacted with tritiated sodium borohydride after treatment with neuraminidase and galactose oxidase. This procedure did not affect the ATPase activity of the enzyme, and all of the covalently bound radioactivity was found in the beta subunit (Mr 54 000). Papain digestion of the tritiated enzyme produced two labeled fragments of Mr 40 000 and 16 000. Further proteolysis generated an Mr 31 000 peptide from the larger fragment. Unlike the tryptic and chymotryptic sites of the alpha subunit, the sites of papain hydrolysis were insensitive to conformations of the (Na+,K+)-ATPase. Determination of the NH2-terminal sequences was used to arrange the fragments within the linear map of the beta chain. Finally, none of the labeled peptides was released from the membrane under nondenaturing conditions. These results are consistent with a model of the beta subunit containing a 40 000-dalton NH2-terminal piece and a 16 000-dalton COOH-terminal piece. Both fragments have extracellularly exposed carbohydrate and at least one membrane-bound domain.     

Palytoxin Acts on Na+,K+-ATPase but not Nongastric H+,K+-ATPase

Palytoxin (PTX) opens a pathway for ions to pass through Na,K-ATPase. We investigate here whether PTX also acts on nongastric H,K-ATPases. The following combinations of cRNA were expressed in Xenopus laevis oocytes: Bufo marinus bladder H,K-ATPase α₂- and Na,K-ATPase β₂-subunits; Bufo Na,K-ATPase α₁- and Na,K-ATPase β₂-subunits; and Bufo Na,K-ATPase β₂-subunit alone. The response to PTX was measured after blocking endogenous Xenopus Na,K-ATPase with 10 μm ouabain. Functional expression was confirmed by measuring ⁸⁶Rb uptake. PTX (5 nm) produced a large increase of membrane conductance in oocytes expressing Bufo Na,K-ATPase, but no significant increase occurred in oocytes expressing Bufo H,K-ATPase or in those injected with Bufo β₂-subunit alone. Expression of the following combinations of cDNA was investigated in HeLa cells: rat colonic H,K-ATPase α₁-subunit and Na,K-ATPase β₁-subunit; rat Na,K-ATPase α₂-subunit and Na,K-ATPase β₂-subunit; and rat Na,K-ATPase β₁- or Na,K-ATPase β₂-subunit alone. Measurement of increases in ⁸⁶Rb uptake confirmed that both rat Na,K and H,K pumps were functional in HeLa cells expressing rat colonic HKα₁/NKβ₁ and NKα₂/NKβ₂. Whole-cell patch-clamp measurements in HeLa cells expressing rat colonic HKα₁/NKβ₁ exposed to 100 nm PTX showed no significant increase of membrane current, and there was no membrane conductance increase in HeLa cells transfected with rat NKβ₁- or rat NKβ₂-subunit alone. However, in HeLa cells expressing rat NKα₂/NKβ₂, outward current was observed after pump activation by 20 mm K⁺ and a large membrane conductance increase occurred after 100 nm PTX. We conclude that nongastric H,K-ATPases are not sensitive to PTX when expressed in these cells, whereas PTX does act on Na,K-ATPase.     

Localization of Na+, K+-ATPase in brain.

Enzyme activity, representing the sites of K+-stimulated p-nitrophenylphosphatase, a component of the sodium, potassium-stimulated-adenosinetriphosphatase system, has been localized in the somatosensory cortex of the rat brain. The reaction product is most obviously associated with fibers that are thought to be axons and dendrites. Large dendrite-like fibers appear to arise in layer 5 of the cortex and arborize in layers 1 through 4. Smaller, reactive fibers are found throughout the cortical layers. Neuron cell bodies did not exhibit substantial enzymatic activity. It did not appear that glia contributed significantly to the activity in cerebral cortex.     

Crystallization patterns of membrane-bound (Na+ + K+)-ATPase

Extensive formation of two-dimensional crystals of the proteins of the pure membrane-bound $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ is induced during prolonged incubation with vanadate and magnesium. Some membrane crystals are formed in medium containing magnesium and phosphate. Computer-averaged images of the two-dimensional crystals show that the unit cell in vanadate-induced crystals contains a protomeric αβ-unit of the enzyme protein. In phosphate-induced crystals an $\text{(αβ)}{}_2\text{-}\text{unit}$ occupies one unit cell suggesting that interactions between αβ-units can be of importance in the function of the Na⁺, K⁺ pump.     

Crystallization patterns of membrane-bound (Na+ +K+)-ATPase

Extensive formation of two-dimensional crystals of the proteins of the pure membrane-bound (Na+ +K+)-ATPase is induced during prolonged incubation with vanadate and magnesium. Some membrane crystals are formed in medium containing magnesium and phosphate. Computer-averaged images of the two-dimensional crystals show that the unit cell in vanadate-induced crystals contains a protomeric alpha beta-unit of the enzyme protein. In phosphate-induced crystals an (alpha beta) 2-unit occupies one unit cell suggesting the interactions between alpha beta-units can be of importance in the function of the Na+, K+ pump.     

Glucagon stimulation of hepatic Na+, K+-ATPase

Summary In the perfused rat liver administration of glucagon was shown to result in a transiently increased uptake of K⁺, indicating the possible involvement of the Na⁺, K⁺-ATPase. Direct measurement of the activity of Na⁺, K⁺-ATPase revealed a two-fold stimulation of the enzyme by glucagon. The effect of glucagon on the activity of the enzyme was immediate. Simultaneously with the increase in the activity of the Na⁺, K⁺-ATPase, the activity of Mg²⁺-ATPase decreased. In order to evaluate whether the activation of the Na⁺, K⁺-ATPase by glucagon is related to the metabolic effects of the hormone, experimental conditions known to interfere with the activity of the enzyme were employed and glucagon stimulation of Ca²⁺-efflux, mitochondrial metabolism and gluconeogenesis were measured. K⁺-free perfusate, high K⁺ perfusate or ouabain interfered to varying degrees with the glucagon stimulation of these responses. The combination of K⁺-free perfusate and ouabain almost completely abolished the glucagon stimulation of all three parameters. These results demonstrate the glucagon stimulation of Na⁺, K⁺-ATPase and raise the possibility that the activation of the enzyme by glucagon might be a necessary link for the manifestation of its metabolic effects.     

Electrogenic ion transport by Na+,K+-ATPase

Experimental data on the ion electrogenic transport by Na+,K+-ATPase available in the literature are analyzed. Special attention is paid to the measurements of unsteady-state electric currents initiated by alternating voltage or rapid introduction of the substrate. In the final part, a physical model of the Na+,K+-ATPase functioning is discussed. According to this model, active transport is carried out by opening and closing of the access channels used for the sodium and potassium exchange between solutions on either side of the membrane. The model explains most of the experimental data, although some details (the channel size, rates of individual transport steps) need further refinement.     

Salt,Na+,K+-ATPase and hypertension

Chronic hypertension is characterized by a persistent increase in vascular tone. Sodium-rich diets promote hypertension; however, the underlying molecular mechanisms are not fully understood. Variations in the sodium content of the diet, through hormonal mediators such as dopamine and angiotensin II, modulate renal tubule Na⁺,K⁺-ATPase activity. Stimulation of Na⁺,K⁺-ATPase activity increases sodium transport across the renal proximal tubule epithelia, promoting Na⁺ retention, whereas inhibited Na⁺,K⁺-ATPase activity decreases sodium transport, and thereby natriuresis. Diets rich in sodium also enhance the release of adrenal endogenous ouabain-like compounds (OLC), which inhibit Na⁺,K⁺-ATPase activity, resulting in increased intracellular Na⁺ and Ca²⁺ concentrations in vascular smooth muscle cells, thus increasing the vascular tone, with a corresponding increase in blood pressure. The mechanisms by which these homeostatic processes are integrated in response to salt intake are complex and not completely elucidated. However, recent scientific findings provide new insights that may offer additional avenues to further explore molecular mechanisms related to normal physiology and pathophysiology of various forms of hypertension (i.e. salt-induced). Consequently, new strategies for the development of improved therapeutics and medical management of hypertension are anticipated.     

Dissociation of Na+,K+-ATPase inhibition from digitalis inotropy.

Recent findings from our laboratory as well as those of other laboratories do not support the postulation that the mechanism of the positive inotropic action of digitalis is due to inhibition of NA,K-ATPase. Using short-acting digitalis steroids and drug washout experiments, in isolated myocardial preparations, it has been demonstrated that Na,K-ATPase isolated from such preparations is still significantly inhibited, whereas the positive inotropic effect is no longer present. Also, based on kinetic measurements the two exponential rate constants observed for drug half-life, a rapid and slow phase, were found to be associated, respectively, with the very short inotropic half-life and the very long enzyme inhibition half-life. In addition, a dissociation of the transient inotropic effects of digitalis was observed from the long lasting cardiotoxic effects of digitalis during drug washout. Moreover, a temporal correlation was noted between the persistent inhibitory effects of digitalis on Na,K-ATPase and the persistent cardiotoxic effects of digitalis. Therefore, it is concluded that inhibition of Na,K-ATPase is not responsible for the positive inotropic action of digitalis, but may be the mechanism, at least in part, for certain cardiotoxic effects of digitalis.     

In search of synaptosomal Na+, K+-ATPase regulators

The arrival of the nerve impulse to the nerve endings leads to a series of events involving the entry of sodium and the exit of potassium. Restoration of ionic equilibria of sodium and potassium through the membrane is carried out by the sodium/potassium pump, that is the enzyme Na⁺,K⁺-ATPase. This is a particle-bound enzyme that concentrates in the nerve ending or synaptosomal membranes. The activity of Na⁺,K⁺-ATPase is essential for the maintenance of numerous reactions, as demonstrated in the isolated synaptosomes. This lends interest to the knowledge of the possible regulatory mechanisms of Na⁺,K⁺-ATPase activity in the synaptic region. The aim of this review is to summarize the results obtained in the author's laboratory, that refer to the effect of neurotransmitters and endogenous substances on Na⁺,K⁺-ATPase activity. Mention is also made of results in the field obtained in other laboratories. Evidence showing that brain Na⁺,K⁺-ATPase activity may be modified by certain neurotransmitters and insulin have been presented. The type of change produced by noradrenaline, dopamine, and serotonin on synaptosomal membrane Na⁺,K⁺-ATPase was found to depend on the presence or absence of a soluble brain fraction. The soluble brain fraction itself was able to stimulate or inhibit the enzyme, an effect that was dependent in turn on the time elapsed between preparation and use of the fraction. The filtration of soluble brain fraction through Sephadex G-50 allowed the separation of two active subfractions: peaks I and II. Peak I increased Na⁺,K⁺- and Mg²⁺-ATPases, and peak II inhibited Na⁺,K⁺-ATPase. Other membrane enzymes such as acetylcholinesterase and 5′-nucleotidase were unchanged by peaks I or II. In normotensive anesthetized rats, water and sodium excretion were not modified by peak I but were increased by peak II, thus resembling ouabain effects.³H-ouabain binding was unchanged by peak I but decreased by peak II in some areas of the CNS assayed by quantitative autoradiography and in synaptosomal membranes assayed by a filtration technique. The effects of peak I and II on Na⁺,K⁺-ATPase were reversed by catecholamines. The extent of Na⁺,K⁺-ATPase inhibition by peak II was dependent on K⁺ concentration, thus suggesting an interference with the K⁺ site of the enzyme. Peak II was able to induce the release of neurotransmitter stored in the synaptic vesicles in a way similar to ouabain. Taking into account that peak II inhibits only Na⁺,K⁺-ATPase, increases diuresis and natriuresis, blocks high affinity³H-ouabain binding, and induces neurotransmitter release, it is suggested that it contains an ouabain-like substance.     

Na+,K+-ATPase: 40 years of investigations

Nobel Prize of 1997 in chemistry was awarded to three scientists fruitfully working in bioenergetics. J. Walker and P. Boyer were awarded the Prize for studies of structure and mechanism of functioning of the H+-transporting (mitochondrial) adenosine triphosphatase. The decision of the Nobel Committee was not unexpected, since these works were very impressive. Special attention was drawn to the fact that the investigations of Walker, the recognized specialist in protein structure, made possible the experimental confirmation of regularities in the mitochondrial ATPase functioning discovered by P. Boyer. The third member of this triumph of bioenergetics is Jens-Christian Skou who described the Na+,K+-activated ATPase in 1957 and then characterized the enzyme properties in detail. Forty years of his scientific biography were devoted to this enzyme. Along with accumulation of scientific knowledge, that constituted the fundamental contribution to bioenergetics (J.Skou is rightfully considered as one of founders of this branch in the present-day biology), the world-wide known school of scientists was established, and starting from 1974, members of this school organize regular conferences on this enzyme.     

Na+, K+-ATPase of the canine mesenteric artery

Na+,K+-ATPase, the enzymatic moiety that operates as the electrogenic sodium-potassium pump of the cell plasma membrane, is inhibited by cardiac glycosides, and this specific interaction of a drug with an enzyme has been considered to be responsible for digitalis-induced vascular smooth muscle contraction. Although studies aimed at localization, isolation, and measurement of the Na+,K+-ATPase activity (or Na+, K- pump activity) indicate its presence in vascular smooth muscle sarcolemma, its characterization as the putative vasopressor receptor site for cardiac glycosides has depended on pharmacological studies of vascular response in vivo and on isolated artery contractile responses in vitro. More recently, radioligand-binding studies using [3H]ouabain have aided in the characterization of drug-enzyme interaction. Such studies indicate that in canine superior mesenteric artery (SMA), Na+,K+-ATPase is the only specific site of interaction of ouabain with resultant inhibition of the enzyme. The characteristics of [3H]ouabain binding to this site are similar to those of purified or partially purified Na+,K+-ATPase of other tissues, which suggests that if Na+,K+-ATPase inhibition is causally related to digitalis-mediated effects on vascular smooth muscle contraction, then therapeutic concentrations of cardiac glycosides could act to cause SMA vasoconstriction. The additional finding from radioligand-binding studies that Na+,K+-ATPase exists in much smaller quantities (density of sites per cell) in SMA than in either heart or kidney may have implications concerning its physiological, biochemical or pharmacological role in modulating vascular muscle tone.     

Na+,K+-ATPase and Ca2+-ATPase isozymes in malignant neoplasms

A current state of researches on mechanisms of ion homeostasis regulation in the specific conditions of the uncontrolled malignant tumor growth (mainly in carcinomas) concerning the contribution of Na+,K+-ATPase, plasma membrane and sarco(endo)plasmic reticulum Ca2+-ATPases has been reviewed. Particular attention has been focused on the molecular and biochemical links providing the redistribution of the transporting ATPases isozyme pattern for the regulatory requirements of the cell signaling pathways at stable proliferation and viability in malignancy.     

C-peptide,Na+,K+-ATPase,and Diabetes

Na⁺,K⁺-ATPase is an ubiquitous membrane enzyme that allows the extrusion of three sodium ions from the cell and two potassium ions from the extracellular fluid. Its activity is decreased in many tissues of streptozotocin-induced diabetic animals. This impairment could be at least partly responsible for the development of diabetic complications. Na⁺,K⁺-ATPase activity is decreased in the red blood cell membranes of type 1 diabetic individuals, irrespective of the degree of diabetic control. It is less impaired or even normal in those of type 2 diabetic patients. The authors have shown that in the red blood cells of type 2 diabetic patients, Na⁺,K⁺-ATPase activity was strongly related to blood C-peptide levels in non–insulin-treated patients (in whom C-peptide concentration reflects that of insulin) as well as in insulin-treated patients. Furthermore, a gene-environment relationship has been observed. The alpha-1 isoform of the enzyme predominant in red blood cells and nerve tissue is encoded by the ATP1A1 gene.Apolymorphism in the intron 1 of this gene is associated with lower enzyme activity in patients with C-peptide deficiency either with type 1 or type 2 diabetes, but not in normal individuals. There are several lines of evidence for a low C-peptide level being responsible for low Na⁺,K⁺-ATPase activity in the red blood cells. Short-term C-peptide infusion to type 1 diabetic patients restores normal Na⁺,K⁺-ATPase activity. Islet transplantation, which restores endogenous C-peptide secretion, enhances Na⁺,K⁺-ATPase activity proportionally to the rise in C-peptide. This C-peptide effect is not indirect. In fact, incubation of diabetic red blood cells with C-peptide at physiological concentration leads to an increase of Na⁺,K⁺-ATPase activity. In isolated proximal tubules of rats or in the medullary thick ascending limb of the kidney, C-peptide stimulates in a dose-dependent manner Na⁺,K⁺-ATPase activity. This impairment in Na⁺,K⁺-ATPase activity, mainly secondary to the lack of C-peptide, plays probably a role in the development of diabetic complications. Arguments have been developed showing that the diabetesinduced decrease in Na⁺,K⁺-ATPase activity compromises microvascular blood flow by two mechanisms: by affecting microvascular regulation and by decreasing red blood cell deformability, which leads to an increase in blood viscosity. C-peptide infusion restores red blood cell deformability and microvascular blood flow concomitantly with Na⁺,K⁺-ATPase activity. The defect in ATPase is strongly related to diabetic neuropathy. Patients with neuropathy have lower ATPase activity than those without. The diabetes-induced impairment in Na⁺,K⁺-ATPase activity is identical in red blood cells and neural tissue. Red blood cell ATPase activity is related to nerve conduction velocity in the peroneal and the tibial nerve of diabetic patients. C-peptide infusion to diabetic rats increases endoneural ATPase activity in rat. Because the defect in Na⁺,K⁺-ATPase activity is also probably involved in the development of diabetic nephropathy and cardiomyopathy, physiological C-peptide infusion could be beneficial for the prevention of diabetic complications.     

Simplified Digitalis-like Compounds acting on Na+, K+-ATPase

Digitalis compounds are used in the treatment of congestive heart failure as positive inotropic agents; their action is mainly due to the inhibition of Na+,K+-ATPase. A well-known drawback is their arrhythmogenic potential. Attempts to find safer digitalis-like compounds by means of molecular simplifications of the typical 5β,14β-steroidal skeleton, which appeared in the medicinal chemistry literature from 1990 until 2002, are briefly reviewed. Several novel achievements were obtained in order to better understand the requisites of the digitalis binding site on Na+, K+-ATPase. Only minor simplification, such as cleavage of the D ring of the digitalis skeleton, could preserve the desired inotropic activity, while highly simplified digitalis-like compounds failed to give sufficiently high inotropic potency, even in the presence of a powerful pharmacophore, such as the O-aminoalkyloxime group.     

Na+,K+-ATPase Na+ Affinity in Rat Skeletal Muscle Fiber Types

Previous studies in expression systems have found different ion activation of the Na⁺/K⁺-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na⁺,K⁺-ATPase activity, and the Na⁺ affinity of Na⁺,K⁺-ATPase was studied in total membranes from rat muscle and purified membranes from muscle with different fiber types. The Na⁺ affinity was higher (K _m lower) in oxidative muscle compared with glycolytic muscle and in purified membranes from oxidative muscle compared with glycolytic muscle. Na⁺,K⁺-ATPase isoform analysis implied that heterodimers containing the β₁ isoform have a higher Na⁺ affinity than heterodimers containing the β₂ isoform. Immunoprecipitation experiments demonstrated that dimers with α₁ are responsible for approximately 36% of the total Na,K-ATPase activity. Selective inhibition of the α₂ isoform with ouabain suggested that heterodimers containing the α₁ isoform have a higher Na⁺ affinity than heterodimers containing the α₂ isoform. The estimated K _m values for Na⁺ are 4.0, 5.5, 7.5 and 13 mM for α₁β₁, α₂β₁, α₁β₂ and α₂β₂, respectively. The affinity differences and isoform distributions imply that the degree of activation of Na⁺,K⁺-ATPase at physiological Na⁺ concentrations differs between muscles (oxidative and glycolytic) and between subcellular membrane domains with different isoform compositions. These differences may have consequences for ion balance across the muscle membrane.