Na,K-ATPase function in alternating electric fields.

Alternating currents affect ion transport processes and ATP splitting through changes in the activation of the membrane Na,K-ATPase. Both processes vary with the frequency, and the effective range includes the environmental 60 Hz. ATP splitting by Na,K-ATPase suspensions decreases for the enzyme under normal conditions, with the maximum effect at 100 Hz. ATP splitting increases when the enzyme activity is lowered to less than half its optimal value by changes in temperature, ouabain concentration, etc. These observations can be explained by the effects of the ionic currents on ion binding at the enzyme activation sites. Such a mechanism could account for the effects of electromagnetic fields on cells, as the transmembrane enzyme can convey the effect of an extracellular signal into the cell via ionic fluxes, and the measured threshold field is within the range of reported biological effects.     

Regulation of Na,K-ATPase function in the lens

The two cell types in the lens, epithelium and fiber, have a very different specific activity of Na,K-ATPase; activity is much higher in the epithelium. However, judged by Western blot, fibers and epithelium express a similar amount of both Na,K-ATPase alpha and beta subunit proteins. Na,K-ATPase protein abundance does not tally with Na,K-ATPase activity. Studies were conducted to examine whether protein synthesis plays a role in maintenance of the high Na,K-ATPase activity in lens epithelium. An increase of cytoplasmic sodium was found to increase Na,K-ATPase protein expression in the epithelium, but not in the fibers. The findings illustrate the ability of lens epithelium to synthesize new Na,K-ATPase protein as a way to boost Na,K-ATPase in response to cell damage or pathological events. Methionine incorporation studies suggested Na,K-ATPase synthesis may also play a role in day to day preservation of high Na,K-ATPase activity. Na,K-ATPase protein in lens epithelial cells appeared to be continually synthesized and degraded. Experiments with cycloheximide suggest that specific activity of Na,K-ATPase in the lens epithelium may depend on the ability of the cells to continuously synthesize fresh Na,K-ATPase proteins. However, other factors such as phosphorylation of Na,K-ATPase alpha subunit may also influence Na,K-ATPase activity. When intact lenses were exposed to the agonist thrombin, Na,K-ATPase activity was diminished, but the response was suppressed by inhibitors of the Src family of non-receptor tyrosine kinases. Thrombin elicited tyrosine phosphorylation of lens epithelium membrane proteins, including a 100 kDa protein band thought to be the Na,K-ATPase alpha 1 subunit. It remains to be determined whether a tyrosine phosphorylation mechanism contributes to the low activity of Na,K-ATPase in lens fibers.     

Na,K-ATPase: Isoform structure,function, and expression

An interesting feature of the Na,K-ATPase is the multiplicity of and isoforms. Three isoforms exist for the subunit, 1, 2, and 3, as well for the subunit, 1, 2, and 3. The functional significance of these isoforms is unknown, but they are expressed in a tissue- and developmental-specific manner. For example, all three isoforms of the subunit are present in the brain, while only 1 is present in kidney and lung, and 2 represents the major isoform in skeletal muscle. Therefore, it is possible that each of these isoforms confers different properties on the Na,K-ATPase which allows effective coupling to the physiological process for which it provides energy in the form of an ion gradient. It is also possible that the multiple isoforms are the result of gene triplication and that each isoform exhibits similar enzymatic properties. In this case, the expression of the triplicated genes would be individually regulated to provide the appropriate amount of Na,K-ATPase to the particular tissue and at specific times of development. While differences are observed in such parameters as Na⁺ affinity and sensitivity to cardiac glycosides, it is not known if these properties play a functional role within the cell.Site-directed mutagenesis has identified amino acid residues in the first extracellular region of the subunit as major determinants in the differential sensitivity to cardiac glycosides. Similar studies have failed to identify residues in the second extracellular region involved in cardiac glycoside inhibition. Further analysis of the enzymatic properties of the enzyme, understanding the regulated expression of the genes, and structure-function studies utilizing site-directed mutagenesis should provide new insights into the enzymatic and physiological roles of the various subunit isoforms of the Na,K-ATPase.     

Na,K-ATPase activity in the myocardial sarcolemma in ischemia

The total time-controlled ischemia (up to 45 min) was studied for its effect on the Na,K-ATPase activity, content of nonesterified fatty acids (NEFA) and intensity of lipid peroxidation (LP) in sarcolemmal (SL) preparations and soluble fractions (SF) from the rat and guinea-pig left ventricles. A strong correlation between Na, K-ATPase inhibition and NEFA accumulation was revealed in the SF. On the contrary, changes in the NEFA content and LP level both in SL and SF did not correlate with a decrease in the enzymic activity. Pretreatment with albumin (0.5 mg/ml) induced equally small increase both in the control and in the ischemic SL preparations. It is suggested that the Na,K-ATPase activity during a short period of myocardial ischemia (up to 45 min) may be due to the NEFA accumulation in the cytosolic and/or extracellular space, but not in SL.     

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⁺.     

Regulatory function of the Na,K-ATPase α2-isoform

A present review is devoted to the analysis of literature data and results of our own research in the field of the Na,K-ATPase molecular diversity. Abundant evidence shows that the Na,K-ATPase α2 isoform is not only involved in various specific cell functions but also affected by different regulatory factors as compared to the α1 isoform which carries the main pump function. Data gathered suggest that these features of α2 isoform are determined by its functional and molecular environment, localization in specific cellular microdomains and also by less stable integration into the cell membrane as compared to other isoforms of the Na,K-ATPase α subunit.     

Increased brain Na, K-ATPase activity of rats under stress

The activities of Na, K- and Mg-dependent ATPases were measured in crude synaptosomal fractions isolated from the rat brain gray matter. Prolonged (6 h) exposure to emotional painful stress stimulated Na, K-ATPase activity by 40% without affecting that of Mg-ATPase. Preliminary injection of the free radical scavenger ionol presented Na, K-ATPase activation, thus suggesting the involvement of lipid peroxidation initiated in brain tissues under stress in acceleration of NA-pump function. However, model studies with lipid peroxidation induced in vitro by an ascorbate-dependent system in a membranous suspension demonstrated an opposite effect, i. e. fast inhibition of Na, K-ATPase. Possible reasons for the different effects of lipid peroxidation in vivo under stress and on Na, K-ATPase activity in vitro are discussed. It is concluded that activation of Na K-ATPase is a mechanism which is responsible for acceleration of reflex conditioning and for the maintenance of the conditioned reflexes in stress-exposed animals.     

Organophosphate inhibition of avian salt gland Na, K-ATPase activity

1. Adult black ducks (Anas rubripes) were given freshwater or saltwater (1.5% NaCl) for 11 days and half of each group was also given an organophosphate (17 p.p.m. fenthion) in the diet on days 6-11. 2. After 11 days, ducks drinking saltwater had lost more weight and had higher plasma Na and uric acid concentrations and osmolalities than birds drinking freshwater. 3. Saltwater treatment stimulated the salt gland to increased weight and Na, K-ATPase activity. 4. Fenthion generally reduced plasma and brain cholinesterase activity and depressed cholinesterase and Na, K-ATPase activities in salt glands of birds drinking saltwater.     

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.     

Regulation of Na, K-ATPase activity by monovalent cations]

A deviation from optimal conditions of the Na, K-ATPase reaction results in a drastic change in the plot: enzyme activity versus Na/K ratio. Acidification of the medium and a decrease in Mg2+ concentration and temperature results in two peaks on the curve at Na/K ratio of about 1 and at Na/K ratio greater than 4. The enhancement of pH of the medium and increase in Mg2+ concentration decreases the first peak and increases the second one. A comparison of these curves for hydrolysis of ATP, UTP and p-nitrophenylphosphate and temperature dependence of the hydrolysis of the substrates suggest that the anomalies observed may be accounted for the Na+ effect on the K-sites or K+ effect on the Na-sites under conditions when cation-binding sites are heterogeneous.     

Factors determining the functional activity of Na,K-ATPase]

A comparative estimation was made of modifications of Na,K-ATPase and Mg-ATPase parameters in the process of phylogenesis and as a result of sudden thermal selection. On the basis of our own and literary data a suggestion was put forward about the availability of quite different ways of thermal adaptation in ATP-hydrolyzing enzymes associated with different physiological functions.     

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.     

Functional activity of tissue Na,K-ATPase in health and in pathology]

The review deals with two basic possibilities of regulation of ion-transporting activity. The first possibility is connected with the changes in the number of working molecules of Na, K-ATP-ase and the second one with the change in the number of turns of active molecules of the pump.     

Changes in the receptor function of Na,K-ATPase during hypoxia and ischemia

Molecular Biology - Na,K-ATPase maintains sodium and potassium homeostasis. It is the only known receptor for cardiotonic steroids such as ouabain. Binding of ouabain to Na,K-ATPase leads to the...     

In vivo modification of Na,K-ATPase activity in Drosophila

We have constructed and characterized transgenic Drosophila lines with modified Na⁺,K⁺-ATPase activity. Using a temperature dependent promoter from the hsp70 gene to drive expression of wild-type α subunit cDNA, we can conditionally rescue bang-sensitive paralysis and ouabain sensitivity of a Drosophila Na⁺,K⁺-ATPase α subunit hypomorphic mutant, 2206. In contrast, a mutant α subunit (α^D369N) leads to increased bang-sensitive paralysis and ouabain sensitivity. We can also generate temperature dependent phenotypes in wild-type Drosophila using the same hsp70 controlled α transgenes. Ouabain sensitivity was as expected, however, both bang sensitive paralysis or locomotor phenotypes became more severe regardless of the type of α subunit transgene. Using the Gal4-UAS system we have limited expression of α transgenes to cell types that normally express a particular Drosophila Na⁺,K⁺-ATPase β (Nervana) subunit isoform (Nrv1 or 2). The Nrv1-Gal4 driver results in lethality while the Nrv2-Gal4 driver shows reduced viability, locomotor function and uncontrolled wing beating. These transgenic lines will be useful for disrupting function in a broad range of cell types.     

Loss of thyroidal inducibility of Na,K-ATPase with neoplastic transformation in tissue culture

Thyroidal induction of the plasma membrane Na,K-ATPase is a characteristic of mammalian tissues that exhibit a thermogenic response to this hormone. To facilitate analysis of the pathways mediating this response, we defined the conditions needed for reproducible thyroidal induction of this enzyme, as well as mitochondrial cytochrome c oxidase, in established cell lines in tissue cultures. In confluent monolayers of nontransformed mouse embryo fibroblasts (C3H/10T1/2), triiodothyronine modulated Na,K-ATPase and cytochrome c oxidase activities in a concentration- and time-dependent manner. Similar increases in Na,K-ATPase activity were obtained in other rodent embryo cells (SWISS/3T3 and NIH/3T3) and in human fibroblasts (WI-38). In contrast, neoplastic transformation of all of these cell lines resulted in loss of inducibility of Na,K-ATPase by thyroid hormone, regardless of the initiating mechanism (i.e. spontaneous, x-ray, chemicals, viruses).     

The role of the substrate structure in the function of Na,K-ATPase

The mechanism of Na,K-ATPase function is reviewed. The peculiarities of hydrolysis of various substrates are described. The experimental results testify to the effect of substrate structure on the E2----E1 transition, rate of Na+ transport, K-dependent phosphatase activation and the quaternary structure of Na,K-ATPase. A conclusion is drawn that the proton-acceptor properties of the substrate play a role in the regulation of ion transport by Na,K-ATPase.     

Hyposmotic stress causes ATP release and stimulates Na,K-ATPase activity in porcine lens

Purinergic receptors in lens epithelium suggest lens function can be altered by chemical signals from aqueous humor or the lens itself. Here we show release of ATP by intact porcine lenses exposed to hyposmotic solution (200 mOsm). 18α-glycyrrhetinic acid (AGA) added together with probenecid eliminated the ATP increase. N-ethylmaleimide (200 μM), an exocytotic inhibitor, had no significant effect on ATP increase. Lenses exposed to hyposmotic solution displayed a ~400% increase of propidium iodide (PI) entry into the epithelium. The increased ability of PI (MW 668) to enter the epithelium suggests possible opening of connexin and/or pannexin hemichannels. This is consistent with detection of connexin 43, connexin 50, and pannexin 1 in the epithelium and the ability of AGA + probenecid to prevent ATP release. Na,K-ATPase activity doubled in the epithelium of lenses exposed to hyposmotic solution. The increase of Na,K-ATPase activity did not occur when apyrase was used to prevent extracellular ATP accumulation or when AGA + probenecid prevented ATP release. The increase of Na,K-ATPase activity was inhibited by the purinergic P2 antagonist reactive blue-2 and pertussis toxin, a G-protein inhibitor, but not by the P2X antagonist PPADS. Hyposmotic solution activated Src family kinase (SFK) in the epithelium, judged by Western blot. The SFK inhibitor PP2 abolished both SFK activation and the Na,K-ATPase activity increase. In summary, hyposmotic shock-induced ATP release is sufficient to activate a purinergic receptor- and SFK-dependent mechanism that stimulates Na,K-ATPase activity. The responses might signify an autoregulatory loop initiated by mechanical stress or osmotic swelling.     

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.     

Cation selectivity of gastric H,K-ATPase and Na,K-ATPase chimeras.

Chimeras of the catalytic subunits of the gastric H,K-ATPase and Na, K-ATPase were constructed and expressed in LLC-PK1 cells. The chimeras included the following: (i) a control, H85N (the first 85 residues comprising the cytoplasmic N terminus of Na,K-ATPase replaced by the analogous region of H,K-ATPase); (ii) H85N/H356-519N (the N-terminal half of the cytoplasmic M4-M5 loop also replaced); and (iii) H519N (the entire front half replaced). The latter two replacements confer a decrease in apparent affinity for extracellular K+. The 356-519 domain and, to a greater extent, the H519N replacement confer increased apparent selectivity for protons relative to Na+ at cytoplasmic sites as shown by the persistence of K+ influx when the proton concentration is increased and the Na+ concentration decreased. The pH and K+ dependence of ouabain-inhibitable ATPase of membranes derived from the transfected cells indicate that the H519N and, to a lesser extent, the H356-519N substitution decrease the effectiveness of K+ to compete for protons at putative cytoplasmic H+ activation sites. Notable pH-independent behavior of H85N/H356-519N at low Na+ suggests that as pH is decreased, Na+/K+ exchange is replaced largely by (Na+ + H+)/K+ exchange. With H519N, the pH and Na+ dependence of pump and ATPase activities suggest relatively active H+/K+ exchange even at neutral pH. Overall, this study provides evidence for important roles in cation selectivity for both the N-terminal half of the M4-M5 loop and the adjacent transmembrane helice(s).