K+Nutrition and Na+Toxicity: The Basis of Cellular K+/Na+Ratios

The capacity of plants to maintain a high cytosolic K⁺/Na⁺ratiois likely to be one of the key determinants of plant salt tolerance.Important progress has been made in recent years regarding theidentification and characterization of genes and transportersthat contribute to the cytosolic K⁺/Na⁺ratio. For K⁺uptake,K⁺efflux and K⁺translocation to the shoot, genes have been isolatedthat encode K⁺uptake and K⁺release ion channels and K⁺carriersthat are coupled to either a H⁺or Na⁺gradient. Although thepicture is less clear for the movement of Na⁺, one pathway,in the form of non-selective ion channels, is likely to playa role in Na⁺uptake, whereas Na⁺efflux and compartmentationare likely to be mediated by H⁺-coupled antiport. In addition,several proteins have been characterized that play prominentroles in the regulation of K⁺and/or Na⁺fluxes. In this BotanicalBriefing we will discuss the functions and interactions of thesegenes and transporters in the broader context of K⁺nutritionand Na⁺toxicity. Copyright 1999 Annals of Botany Company Salinty, K⁺/N⁺ratio, transporter, membrane.     

Inhibitors of the Na+ K+-atpase

1. The major ionmotive ATPase, in animal cells, is the Na⁺, K⁺-ATPase or sodium pump.2. This membrane bound enzyme is responsible for the translocation of Na⁺ ions and K⁺ ions across the plasma membrane, an active transport mechanism that requires the expenditure of the metabolic energy stored within the ATP molecule.3. This ubiquitous enzyme controls directly or indirectly many essential cellular functions, such as, cell volume, free calcium concentration and membrane potential.4. It is, therefore, apparent that alterations in its regulation may play key roles in pathological processes.     

The effect of Na+ and K+ on the thermal denaturation of Na+ and + K+-dependent ATPase.

To increase our understanding of the physical nature of the Na+ and K+ forms of the Na+ + K+-dependent ATPase, thermal-denaturation studies were conducted in different types of ionic media. Thermal-denaturation measurements were performed by measuring the regeneration of ATPase activity after slow pulse exposure to elevated temperatures. Two types of experiments were performed. First, the dependence of the thermal-denaturation rate on Na+ and K+ concentrations was examined. It was found that both cations stabilized the pump protein. Also, K+ was a more effective stabilizer of the native state than was Na+. Secondly, a set of thermodynamic parameters was obtained by measuring the temperature-dependence of the thermal-denaturation rate under three ionic conditions: 60 mM-K+, 150 mM-Na+ and no Na+ or K+. It was found that ion-mediated stabilization of the pump protein was accompanied by substantial increases in activation enthalpy and entropy, the net effect being a less-pronounced increase in activation free energy.     

Inward-directed current generated by the Na+,K+ pump in Na+- and K+-free medium

In Na⁺- and K⁺-free solution, an inward-directed current can be detected in Xenopus oocytes, which is inhibited by cardic glycosides and activated by ATP. Therefore, it is assumed to be generated by the Na⁺, K⁺ pump. At negative membrane potentials, the pump current increases with more negative potentials and with increasing [H⁺] in the external medium. This current is not observed when Mg²⁺ instead of Ba²⁺ is the only divalent cation present in the bath medium, and it does not depend on whether Na⁺ or K⁺ is present internally. At 5 to 10 mM Na⁺ externally, maximum pump-generated current is obtained while no current can be detected in presence of physiological [Na⁺]. It is suggested that in low-Na⁺ and K⁺-free medium the Na⁺, K⁺ pump molecule can either form a conductive pathway that is permeable to Ba²⁺ or protons or operate in its conventional transport mode accepting Ba²⁺ as a K⁺ congener. A reversed pump mode or an electrogenic uncoupled Na⁺-efflux mode is excluded.     

K+-independent active transport of Na+ by the (Na+ and K+)-stimulated adenosine triphosphatase

The (Na+ and K+)-stimulated adenosine triphosphatase (Na+,K+)-ATPase) from canine kidney reconstituted into phospholipid vesicles showed an ATP-dependent, ouabain-inhibited uptake of 22Na+ in the absence of added K+. This transport occurred against a Na+ concentration gradient, was not affected by increasing the K+ concentration to 10 microM (four times the endogenous level), and could not be explained in terms of Na+in in equilibrium Na+out exchange. K+-independent transport occurred with a stoichiometry of 0.5 mol of Na+ per mol of ATP hydrolyzed as compared with 2.9 mol of Na+ per mol of ATP for K+-dependent transport.     

A reconstituted Na+ + K+ pump in liposomes containing purified (Na+ + K+)-ATPase from kidney medulla.

Liposomes containing either purified or microsomal (Na+,K+)-ATPase preparations from lamb kidney medulla catalyzed ATP-dependent transport of Na+ and K+ with a ratio of approximately 3Na+ to 2K+, which was inhibited by ouabain. Similar results were obtained with liposomes containing a partially purified (Na+,K+)-ATPase from cardiac muscle. This contrasts with an earlier report by Goldin and Tong (J. Biol. Chem. 249, 5907-5915, 1974), in which liposomes containing purified dog kidney (Na+,K+)-ATPase did not transport K+ but catalyzed ATP-dependent symport of Na+ and Cl-. When purified by our procedure, dog kidney (Na+,K+)-ATPase showed some ability to transport K+ but the ratio of Na+ : K+ was 5 : 1.     

Activation of (Na+ + K+)-ATPase

Enzymes catalyze essential chemical reactions needed for living processes. (Na+ +K+)-ATPase (NKA) is one of the key enzymes that control intracellular ion homeostasis and regulate cardiac function. Little is known about activation of NKA and its biological impact. Here we show that native activity of NKA is markedly elevated when protein-protein interaction occurs at the extracellular DVEDSYGQQWTYEQR (D-R) region in the alpha-subunit of the enzyme. The apparent catalytic turnover of NKA is approximately twice as fast as the controls for both ouabain-resistant and ouabain-sensitive enzymes. Activation of NKA not only markedly protects enzyme function against denaturing, but also directly affects cellular activities by regulating intracellular Ca2+ transients and inducing a positive inotropic effect in isolated rat cardiac myocytes. Immunofluorescent labeling indicates that the D-R region of NKA is not a conventional digitalis-binding site. Our findings uncover a novel activation site of NKA that is capable of promoting the catalytic function of the enzyme and establish a new concept that activating of NKA mediates cardiac contraction.     

Charge translocation by the Na+/K+ pump under Na+/Na+ exchange conditions: intracellular Na+ dependence

The effect of intracellular (i) and extracellular (o) Na+ on pre-steady-state transient current associated with Na+/Na+ exchange by the Na+/K+ pump was investigated in the vegetal pole of Xenopus oocytes. Current records in response to 40-ms voltage pulses from -180 to +100 mV in the absence of external Na+ were subtracted from current records obtained under Na+/Na+ exchange conditions. Na+-sensitive transient current and dihydroouabain-sensitive current were equivalent. The quantity of charge moved (Q) and the relaxation rate coefficient (ktot) of the slow component of the Nao+-sensitive transient current were measured for steps to various voltages (V). The data were analyzed using a four-state kinetic model describing the Na+ binding, occlusion, conformational change, and release steps of the transport cycle. The apparent valence of the Q vs. V relationship was near 1.0 for all experimental conditions. When extracellular Na+ was halved, the midpoint voltage of the charge distribution (Vq) shifted -25.3+/-0.4 mV, which can be accounted for by the presence of an extracellular ion-well having a dielectric distance delta=0.69+/-0.01. The effect of changes of Nai+ on Nao+-sensitive transient current was investigated. The midpoint voltage (Vq) of the charge distribution curve was not affected over the Nao+ concentration range 3.13-50 mM. As Nai+ was decreased, the amount of charge measured and its relaxation rate coefficient decreased with an apparent Km of 3.2+/-0.2 mM. The effects of lowering Nai+ on pre-steady-state transient current can be accounted for by decreasing the charge available to participate in the fast extracellular Na+ release steps, by a slowly equilibrating (phosphorylation/occlusion) step intervening between intracellular Na+ binding and extracellular Na+ release.     

Low-affinity Na+ sites on (Na+ +K+)-ATPase modulate inhibition of Na+-ATPase activity by vanadate

Na+-ATPase activity is extremely sensitive to inhibition by vanadate at low Na+ concentrations where Na+ occupies only high-affinity activation sites. Na+ occupies low-affinity activation sites to reverse inhibition of Na+-ATPase and (Na+, K+)-ATPase activities by vanadate. This effect of Na+ is competitive with respect to both vanadate and Mg2+. The apparent affinity of the enzyme for vanadate is markedly increased by K+. The principal effect of K+ may be to displace Na+ from the low-affinity sites at which it activates Na+-ATPase activity.     

Characteristics of cooperative binding of Na+ and K+ with Na+,K+-ATPase depending upon its oligomeric structure

It has been shown that the desensibilization of the enzymic preparations of Na+, K+-ATPase by urea, DS-Na, digitonin and CHAPS reduces differently the amount of alpha beta-protomer in the enzymic preparations and the Hill coefficients of Na+ and K+. The factors (urea, DS-Na) which cause a more pronounced decrease in the amount of beta-protomer reduce the nH of Na+ for Na+, K+-ATPase and nH of K+ for Na+, K+-ATPase and K+-pNPPase to unit. The analysis of the effects of ATP and pNPP indicates that ATP has a protective effect only in the case of urea and DS-Na, but this effect is not exerted by pNPP (nonallosteric substrate). A conclusion is drawn that cooperative interactions of Na+, K+-ATPase from the brain with Na+ require more higher level of the oligomeric structure of enzyme than cooperative interactions with K+. At the same time these cooperative interactions in the both cases need subunits interactions in the protomer and interactions between cation sites with relatively high affinity.     

Kinetic studies on Na+/K+-ATPase and inhibition of Na+/K+-ATPase by ATP

Na+/K+-ATPase (EC 3.6.1.3) is an important membrane-bound enzyme. In this paper, kinetic studies on Na+/K+-ATPase were carried out under mimetic physiological conditions. By using microcalorimeter, a thermokinetic method was employed for the first time. Compared with other methods, it provided accurate measurements of not only thermodynamic data (deltarHm) but also the kinetic data (Km and Vmax). At 310.15K and pH 7.4, the molar reaction enthalpy (deltarHm) was measured as -40.514 +/- 0.9kJmol(-1). The Michaelis constant (Km) was determined to be 0.479 +/- 0.020 mM and consistent with literature data. The reliability of the thermokinetic method was further confirmed by colorimetric studies. Furthermore, a simple and reliable kinetic procedure was presented for ascertaining the true substrate for Na+/K+-ATPase and determining the effect of free ATP. Results showed that the MgATP complex was the real substrate with a Km value of about 0.5mM and free ATP was a competitive inhibitor with a Ki value of 0.253 mM.     

Kinetics of (Na+ + K+)-ATPase: analysis of the influence of Na+ and K+ by steady-state kinetics

The influence of Na+ and K+ on the steady-state kinetics at 37 degrees C of (Na+ + K+)-ATPase was investigated. From an analysis of the dependence of slopes and intercepts (from double-reciprocal plots or from Hanes plots) of the primary data on Na+ and K+ concentrations a detailed model for the interaction of the cations with the individual steps in the mechanism may be inferred and a set of intrinsic (i.e. cation independent) rate constants and cation dissociation constants are obtained. A comparison of the rate constants with those obtained from an analogous analysis of Na+-ATPase kinetics (preceding paper) provides evidence that the ATP hydrolysis proceeds through a series of intermediates, all of which are kinetically different from those responsible for the Na+-ATPase activity. The complete model for the enzyme thus involves two distinct, but doubly connected, hydrolysis cycles. The model derived for (Na+ + K+)-ATPase has the following properties: The empty, substrate free, enzyme form is the K+-bound form E2K. Na+ (Kd = 9 mM) and MgATP (Kd = 0.48 mM), in that order, must be bound to it in order to effect K+ release. Thus Na+ and K+ are simultaneously present on the enzyme in part of the reaction cycle. Each enzyme unit has three equivalent and independent Na+ sites. K+ binding to high-affinity sites (Kd = 1.4 mM) on the presumed phosphorylated intermediate is preceded by release of Na+ from low-affinity sites (Kd = 430 mM). The stoichiometry is variable, and may be Na:K:ATP = 3:2:1. To the extent that the transport properties of the enzyme are reflected in the kinetic ATPase model, these properties are in accord with one of the models shown by Sachs ((1980) J. Physiol. 302, 219-240) to give a quantitative fit of transport data for red blood cells.     

Interactions of K+ ATP channel blockers with Na+/K+-ATPase

Two K⁺ _ATP channel blockers, 5-hydroxydecanoate (5-HD) and glyburide, are often used to study cross-talk between Na⁺/K⁺-ATPase and these channels. The aim of this work was to characterize the effects of these blockers on purified Na⁺/K⁺-ATPase as an aid to appropriate use of these drugs in studies on this cross-talk. In contrast to known dual effects (activating and inhibitory) of other fatty acids on Na⁺/K⁺-ATPase, 5-HD only inhibited the enzyme at concentrations exceeding those that block mitochondrial K⁺ _ATP channels. 5-HD did not affect the ouabain sensitivity of Na⁺/K⁺-ATPase. Glyburide had both activating and inhibitory effects on Na⁺/K⁺-ATPase at concentrations used to block plasma membrane K⁺ _ATP channels. The findings justify the use of 5-HD as specific mitochondrial channel blocker in studies on the relation of this channel to Na⁺/K⁺-ATPase, but question the use of glyburide as a specific blocker of plasma membrane K⁺ _ATP channels, when the relation of this channel to Na⁺/K⁺-ATPase is being studied.     

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.     

Mathematical modelling of ATP, K+ and Na+ interactions with (Na+ + K+)-ATPase occurring under equilibrium conditions.

The controlling effect of ATP, K+ and Na+ on the rate of (Na+ + K+)-ATPase inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) is used for the mathematical modelling of the interaction of the effectors with the enzyme under equilibrium conditions. 1. Of a series of conceivable interaction models, designed without conceptual restrictions to describe the effector control of inactivation kinetics, only one fits the experimental data described in a preceding paper. 2. The model is characterized by the coexistence of two binding sites for ATP and the coexistence of two separate binding sites for K+ and Na+ on the enzyme-ATP complex. On the basis of this model, the effector parameters fitting the experimental data most closely are estimated by means of nonlinear least-squares fits. 3. The apparent dissociation constants for ATP fo the enzyme-ATP complex or of the enzyme-(ATP)2 complex are computed to lie near 0.0024 mM and 0.34 mM, respectively, irrespective of whether K+ and Na+ were absent or K+ and K+ plus Na+, respectively, were present in the experiments. 4. The origin of the high and the low affinity site for binding of ATP to the (Na+ + K+)-ATPase molecule is traced back to the coexistence of two catalytic centres which, although primarily equivalent as to the reactivity of their thiol groups with NBD-C1, are induced into anticooperative communication by ATP binding and thus show an induced geometric asymmetry. 5. On the basis of the interaction model outlined under item 2 the apparent dissociation constant for K+ or Na+ in the (K+ + Na+)-liganded enzyme-ATP complex are computed to be 1.7 mM and 3.5 mM, respectively. 6. The conclusions concerning the coexistence of two primarily equivalent but anticooperatively interacting catalytic centres and the coexistence of two separate ionophoric centres for Na+ and K+ correspond to the appropriate basic postulates of the flip-flop concept of (Na+ + K+)-ATPase mechanism.     

Dietary cholesterol alters Na+/K+ selectivity at intracellular Na+/K+ pump sites in cardiac myocytes

A modest diet-induced increase in serum cholesterol in rabbits increases the sensitivity of the sarcolemmal Na⁺/K⁺ pump to intracellular Na⁺, whereas a large increase in cholesterol levels decreases the sensitivity to Na⁺. To examine the mechanisms, we isolated cardiac myocytes from controls and from rabbits with diet-induced increases in serum cholesterol. The myocytes were voltage clamped with the use of patch pipettes that contained osmotically balanced solutions with Na⁺ in a concentration of 10 mM and K⁺ in concentrations ([K⁺]_pip) ranging from 0 to 140 mM. There was no effect of dietary cholesterol on electrogenic Na⁺/K⁺ current (I_p) when pipette solutions were K⁺ free. A modest increase in serum cholesterol caused a [K⁺]_pip-dependent increase in I_p, whereas a large increase caused a [K⁺]_pip-dependent decrease in I_p. Modeling suggested that pump stimulation with a modest increase in serum cholesterol can be explained by a decrease in the microscopic association constant K_K describing the backward reaction E₁ + 2K⁺ E₂(K⁺)₂, whereas pump inhibition with a large increase in serum cholesterol can be explained by an increase in K_K. Because hypercholesterolemia upregulates angiotensin II receptors and because angiotensin II regulates the Na⁺/K⁺ pump in cardiac myocytes in a [K⁺]_pip-dependent manner, we blocked angiotensin synthesis or angiotensin II receptors in vivo in cholesterol-fed rabbits. This abolished cholesterol-induced pump inhibition. Because the -isoform of protein kinase C (PKC) mediates effects of angiotensin II on the pump, we included specific PKC-blocking peptide in patch pipette filling solutions. The peptide reversed cholesterol-induced pump inhibition. partial reactions; protein kinase C; angiotensin converting enzyme inhibitors; arteriosclerosis; insulin resistance