©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Influence of Intramembrane Electric Charge on Na,K-ATPase (*)

(Received for publication, July 7, 1994; and in revised form, November 23, 1994)

Irena Klodos (§) Natalya U. Fedosova (¶) Liselotte Plesner

From the Institute of Biophysics, University of Aarhus, DK-8000 Aarhus C, Denmark

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Effects of lipophilic ions, tetraphenylphosphonium (TPP) and tetraphenylboron (TPB), on interactions of Na and K with Na,K-ATPase were studied with membrane-bound enzyme from bovine brain, pig kidney, and shark rectal gland.

Na and K interactions with the inward-facing binding sites, monitored by eosin fluorescence and phosphorylation, were not influenced by lipophilic ions.

Phosphoenzyme interactions with extracellular cations were evaluated through K-, ADP-, and Na-dependent dephosphorylation. TPP decreased: 1) the rate of transition of ADP-insensitive to ADP-sensitive phosphoenzyme, 2) the K affinity and the rate coefficient for dephosphorylation of the K-sensitive phosphoenzyme, 3) the Na affinity and the rate coefficient for Na-dependent dephosphorylation. Pre-steady state phosphorylation experiments indicate that the subsequent occlusion of extracellular cations was prevented by TPP. TPB had opposite effects.

Effects of lipophilic ions on the transition between phosphoenzymes were significantly diminished when Na was replaced by N-methyl-D-glucamine or Tris, but were unaffected by the replacement of Cl by other anions.

Lipophilic ions affected Na-ATPase, Na,K-ATPase, and p-nitrophenylphosphatase activities in accordance with their effects on the partial reactions.

Effects of lipophilic ions appear to be due to their charge indicating that Na and K access to their extracellular binding sites is modified by the intramembrane electric field.


INTRODUCTION

Lipophilic ions like tetraphenylphosphonium (TPP) (^1)and tetraphenylboron (TPB) partition into cell membranes (Andersen et al., 1978; Flewelling and Hubbell, 1986a, 1986b). The ions are located a few angstroms below the membrane surface (Andersen et al., 1978), and they modify the electrostatic potential profile inside the membrane dielectric (Andersen et al., 1978; Flewelling and Hubbell, 1986a; 1986b). It has been shown by Bühler et al.(1991), Läuger (1991a), and Stürmer et al.(1991) that these ions affect the reactivity of membrane-bound Na,K-ATPase toward Na and K. The authors concluded from experiments with fluorescent, potential-sensitive aminostyrylpyridinium dyes that TPP and TPB affect the release of Na from and the binding of K to external sites of Na,K-ATPase. The authors therefore suggested that the external cation binding sites are formed as deep wells, i.e. K binds to and Na is released from ``sites that are located inside the membrane dielectric'' (Läuger, 1991a). Since the binding of Na at the cytoplasmic side of Na,K-ATPase caused relatively small changes in the fluorescence response, the authors concluded that the cytoplasmic sites are located close to the lipid/water interface (Läuger, 1991a).

In the present work we applied biochemical techniques supported by measurements of conformational changes by eosin fluorescence to describe which partial reactions in the Na,K-ATPase reaction cycle are affected by lipophilic ions. The studies were performed with broken membrane Na,K-ATPase preparations; thus, the lipophilic ions had access to both sides of the membrane and their distribution in the membrane was not modified by a trans-membrane potential. To ease the understanding of the experimental design, the following reaction scheme is used as a frame of reference:

In this very simplified scheme, E(1) denotes the form of Na,K-ATPase characterized by a high affinity to ATP and Na, while E(2) denotes the form with a high affinity to K but a relatively low affinity to ATP and Na (Glynn, 1985; Läuger, 1991b). The lower row of the scheme shows reaction of Na and K with the cytoplasmic sites, while the upper row illustrates reaction of cations with the extracellular sites. The phosphoenzyme E(1)P(Na) has m occluded Na ions and reacts with ADP resynthesizing ATP. E(2)P binds K with a very high affinity, dephosphorylates, and forms an intermediate with n occluded K-ions, E(2)(K).

The effect of lipophilic ions on the following partial reactions were studied: transitions between E(2)(K) and E(1)Na were characterized using eosin fluorescence as an indicator of conformation (Skou and Esmann, 1983); the steps leading to formation of phosphoenzymes were studied in experiments where the pre-steady state time course of EP formation was measured, and the individual phosphoenzyme forms were characterized in chase experiments with ADP or K. The entire reaction cycle was characterized through measurements of the steady state (1) phosphorylation level, (2) distribution of ADP- and K-sensitive phosphointermediates, (3) ATP and pNPP hydrolysis activity at varying concentrations of Na or K or both. The effects of lipophilic ions on the overall reaction are compared to their effects on the partial reactions. Preliminary results have been published (Klodos & Plesner, 1992).


MATERIALS AND METHODS

Enzyme Preparation

Na,K-ATPase from bovine brain was prepared according to Klodos et al.(1975). Na,K-ATPase from pig kidney was prepared according to Jørgensen(1974) as modified by Jensen et al.(1984). Na,K-ATPase from shark rectal gland was prepared as described by Skou and Esmann(1979).

The Na,K-ATPase activity of bovine brain, pig kidney, and shark rectal gland enzymes, measured at 37 °C and under standard conditions (Ottolenghi, 1975, but without bovine serum albumin) were 4-5, 27, and 25 unitsbullet(mg of protein), respectively. The maximum phosphorylation site concentrations, measured as described (Klodos et al., 1981), were 0.45-0.55 nmolbullet(mg of protein) for the brain enzyme, 2.7 nmolbullet(mg of protein) for the pig kidney enzyme, and 2.5 nmolbullet(mg of protein) for shark rectal gland Na,K-ATPase.

The protein amount was determined according to Lowry et al. (1951), as described by Jensen and Ottolenghi (1983a), using bovine serum albumin as standard.

Measurement of Eosin Fluorescence

The experiments were performed in a SPEX Fluorolog-2 spectrofluorometer at 20 °C, the excitation wavelength was 530 nm, the slit was 0.1 nm, and the light path was 1 cm. A Schott RG550 filter was used as emission cut-off filter.

The fluorescence response of 0.5 µM eosin to binding of K and subsequently to addition of Na was measured in 10 mM HEPES, 10 mM MES, 10 mM EDTA buffer, pH 7.5, which favors a form of Na,K-ATPase with high affinity to eosin. The pH was adjusted with N-methyl-D-glucamine. The fluorescence of unspecifically bound eosin was measured in the presence of 375 µM ADP, which prevents specific binding of eosin (Esmann, 1992).

TPPCl and NaTPB dissolved in dimethyl sulfoxide were added to final concentrations of 300 and 50 µM, respectively (dimethyl sulfoxide = 0.05%). The pig kidney Na,K-ATPase (30 µg of proteinbulletml) in 2 ml of buffer solution was thermostated and continually stirred. All additions were from hand-held Hamilton syringes. For further analysis, data were expressed in percent of the starting level of fluorescence in each individual experiment.

Phosphorylation-Dephosphorylation Experiments

The experiments were performed at 0 °C and pH 7.4 with 300 µg of proteinbulletml. TPP and TPB, when present, were added from freshly prepared solutions in water at least 10 min prior to the start of the phosphorylation. The phosphorylation medium also contained 0.1 mM MgCl(2), 30 mM imidazole buffer, pH 7.4, at 0 °C and varying [NaCl]. The phosphorylation was started by the addition of 25 µM [-P]ATP. After varying periods of time, the reaction was either stopped by addition of 10% trichloroacetic acid (final concentration 5%) or dephosphorylation was initiated by the addition of a chase. The chase contained NaCl, buffer, and MgCl(2) in the same concentrations as the phosphorylation medium and, in addition, either unlabeled ATP (final concentration 1 mM), unlabeled ATP and KCl (final concentrations were 1 mM and usually 20 mM respectively), or unlabeled ATP and ADP (final concentrations 1 and 2.5 mM). The dephosphorylation was stopped by addition of 10% trichloroacetic acid (final concentration 5%) at the times indicated. The amount of acid-stabile EP was determined according to Klodos et al.(1981), and the results are presented in the figures after subtraction of EP levels, obtained after dephosphorylation for 5 min. The values in the figures are the mean of at least three experiments ± S.E.

Measurement of ATPase Activity

The [-P]ATP hydrolysis was measured in 30 mM histidine buffer, pH 7.4, at 37 °C in the presence of 3 mM ATP, 3 mM MgCl(2), and [NaCl] and [KCl] given in the figures. P(i) was determined according to Lindberg and Ernster(1956).

Measurement of pNPP Hydrolysis

The pNPPase activity was measured according to Ottolenghi(1975), but without bovine serum albumin in the incubation medium. The buffer was 30 mM imidazole buffer, pH 7.5, at 37 °C. The reaction medium contained 20 mM MgCl(2) and 10 mM pNPP. The pNPPase activity was measured as function of [KCl] at a constant salt concentration of the medium, 150 mM, replacing KCl with choline chloride, NMGCl, or NaCl. Identical results were obtained with choline chloride and NMGCl. The experiments in the presence of both KCl and NaCl were performed with or without 100 µM ATP. The amount of the released p-nitrophenol was determined from the optical density at 410 nm.

Data Processing

The data were analyzed using the computer program ``Plot 5.31'' written by Bliss Forbush III, Dept. of Cellular and Molecular Physiology, School of Medicine, Yale University, applying a linear or nonlinear least squares analysis.

Reagents

ATP and ADP were purchased as sodium salts from Boehringer Mannheim, Germany. ATP used in the phosphorylation experiments was converted to its Tris salt by chromatography on a Dowex 1 column (from Sigma), and [-P]ATP was purified on DEAE-Sephadex G-25 (Nørby and Jensen, 1971). ADP was purified by chromatography on a Dowex 50W H column, and the eluate was adjusted to pH 7.1 at 20 °C with 2-amino-2-methyl-1,3-propanediol. p-Nitrophenyl phosphate (pNPP) purchased as sodium salt from Merck, Darmstadt, Germany, was purified by chromatography on Dowex 50W H. The eluate was adjusted with Tris to pH 7.4 at 37 °C. N-Methyl-D-glucamine (NMG) was purchased from Sigma. Eosin was obtained from Gurt, Chadwell Heath, Essex, UK. TPPCl and NaTPB were gifts from Dr. H.-J. Apell, Universität Konstanz, or purchased from Sigma. TPPCl and NaTPB were dissolved in dimethyl sulfoxide or in water. The water solutions of TPPCl and NaTPB were prepared on the day of the experiment. All other reagents were reagent grade.


RESULTS

The description of the results follows the reaction : binding of Na and K to the dephosphoenzyme, phosphorylation, characterization of phosphointermediates, Na,K-ATPase and pNPPase activities, and, finally, a series of experiments aiming at clarification whether the effects of lipophilic ions are due to their charge. It should be noted that: 1) because of its higher partitioning into the lipid phase (Flewelling and Hubbell, 1986a, 1986b) TPB was used in lower concentrations than that of TPP, and 2) whenever K was present only the effect of TPP was tested, as TPB is a strong chelator of K (Flaschka and Barnard, 1960).

Interconversions between Dephosphoenzyme Forms: E(1)Na(m) E(2)(K(n)) Transition

Conformational transitions of the Na,K-ATPase following cation binding to nonphosphorylated enzyme forms were monitored by eosin fluorescence according to Skou and Esmann(1983), who showed that the fluorescence of eosin specifically bound to the enzyme is higher than the fluorescence of eosin in the solution or of nonspecifically bound eosin. Specifically bound eosin was released from its high affinity binding site by the addition of ADP (Esmann, 1992). 375 µM ADP used in the reported experiments was more than sufficient to prevent specific eosin binding. The fluorescence level observed in the presence of ADP was equal to that measured in the presence of saturating K, where E(2)(K(n)), which did not bind eosin specifically, was the only form present. Neither TPP nor TPB had any effect on the affinity of specific eosin binding measured as equilibrium eosin binding (not shown). Both ions affected eosin fluorescence: TPP caused an increase in the fluorescence of nonspecifically bound eosin, i.e. it increased the background fluorescence, while TPB increased the quantum yield of the fluorescence only of specifically bound eosin (Fedosova and Jensen, 1994).

In our experiments, specific eosin binding was induced by buffer (10 mM HEPES + 10 mM MES + 10 mM EDTA, pH 7.5 at 20 °C). The subsequent addition of 4 mM Na did not result in an increase in fluorescence, indicating that already in the absence of Na the specific eosin binding was maximal. The difference between the level of fluorescence in the presence of buffer, Na, or both, and the level in the presence of ADP was equal to the maximal fluorescence increase caused by the eosin binding to the enzyme (DeltaF(max)). DeltaF(max) corresponded therefore to the complete transition of the enzyme from eosin-bound E(1) to the eosin-free E(2) form.

In the absence of Na, the addition of K induced a decay of specific fluorescence which could be fitted adequately with a monoexponential function (Fig. 1, inset). The rate coefficient of the decay (k) and the equilibrium fluorescence decrease (DeltaF/F(0)) were derived. DeltaF/F(0) versus [K] was found to be a hyperbola: DeltaF/F(0) = (DeltaF(max)/F(0))/(1 + K(K)/[K]), where K(K) = 5.2 ± 0.9 µM (not shown). Similar values were previously found in different types of experiments (cf. Esmann, 1992). The rate coefficient of fluorescence decrease, k, in the same range of potassium concentrations, showed a linear dependence on [K] (Fig. 1A) indicating a low affinity K binding.


Figure 1: The effect of K concentrations on the rate coefficient of E(1) E(2) conversion. Inset in panel A shows a typical recording of eosin fluorescence in an experiment performed with pig kidney Na,K-ATPase in the presence of buffer. Ligand additions are shown and fluorescence is expressed as percentage of the initial level. Thick line shows monoexponential fit of the fluorescence decrease. The calculated rate coefficients for the KCl-dependent fluorescence decrease are shown in panels A and B: panel A, without NaCl; panel B, with 4 mM NaCl in the absence (circles) or in the presence of 300 µM TPP (squares). The values are the mean of three experiments ± S.E. Note the 100-fold difference in concentrations between panel A and panel B.



Thus, we observed the same contradiction between affinities estimated from equilibrium and transient experiments as described previously by Karlish et al.(1978) and Glynn and Karlish(1982), who proposed the following scheme to explain this discrepancy:

They assumed that a low affinity K binding to the E(1) form is followed by a conformational transition, poised heavily in favor of the E(2) form (cf. reviews by Glynn(1985) and Glynn and Karlish (1990)). The rate coefficient of disappearance of the E(1) form upon K addition is equal to k = k + k/(1 + K/[K]) and becomes k = k + kbullet[K]/K for [K] K. Glynn et al. (1987) showed that the time course of deocclusion and the conformational change are closely correlated. Thus, k in the model is the rate constant for K deocclusion from the intracellular sites.

In our experiments, the rate coefficient of the specific eosin fluorescence decrease in response to K, k, displayed a linear dependence on [K] (Fig. 1A). The rate constant k for the E(2) E(1) transition was estimated to be 0.03 ± 0.012 s by extrapolation of the straight line to 0 [K]. It is in agreement with the data on deocclusion and conformational transition rate constants measured by different techniques (Glynn, 1985; Glynn et al., 1987).

Since K = K(K)bullet(k/k), and as k is about 300 s (Steinberg and Karlish, 1989) and k = 0.03 s, an approximate value of the dissociation constant for K, K, was estimated to be in the millimolar range.

When 4 mM Na was present, i.e. the starting point was E(1)Na(m), K addition was also followed by a monoexponential decay. The rate constant k for the K deocclusion was unchanged, but the slope of k versus [K] was decreased by a factor of 80 (Fig. 1B) and the K value obtained from equilibrium measurements was 421 µM (Fig. 2). The simplest reaction scheme compatible with these results is


Figure 2: Equilibrium fluorescence change as function of K in the presence of 4 mM NaCl. Eosin fluorescence was measured in 4 mM NaCl, 10 mM HEPES, 10 mM MES, 10 mM EDTA, pH 7.5, and different concentrations of K in the absence (circles) or in the presence of 300 µM TPP (squares). Eosin fluorescence upon addition of K is expressed as percentage of the starting level of fluorescence. The values are the mean of three experiments ± S.E. The curve is a fit of the experimental data to the equation: DeltaF/F(0) = (DeltaF(max)/F(0))/(1 + K/[K]), as described under ``Materials and Methods.'' The K = 421 ± 0.9 µM.



In this model, Na and K compete for the binding to the E(1) form, but neither the K occlusion, defined as E(1)K to E(2)K transition, nor deocclusion are affected by Na. TPP had no effect on the K binding in the presence of Na (Fig. 1B and Fig. 2).

The experiments on Na binding in the absence of K were performed at low buffer concentration (1 mM HEPES + 1 mM MES) where no specific eosin binding was observed. It has been previously shown that in low ionic strength medium the enzyme has the characteristics of an E(2) conformation (Skou and Esmann, 1980; Glynn and Richards, 1982; Jensen and Ottolenghi, 1983b; Klodos and Ottolenghi, 1985). Na addition to the medium was followed by a fluorescence increase which reflected the formation of the sodium-bound form of the enzyme. Neither TPP nor TPB affected the Na dependence of the specific fluorescence increase (not shown). Thus, TPP affected neither Na nor K binding nor K deocclusion.

Formation of Phosphoenzyme

The amount of EP was measured after a 2-s phosphorylation period at NaCl concentrations varying from 0 to 100 mM. We reported previously a marginal effect of the lipophilic ions on the formation of phosphoenzyme under these conditions ( Fig. 2in Klodos and Plesner(1992)).

When the ionic strength was kept constant with NMGCl or TrisCl, no effect of lipophilic ions on the formation of EP was observed (Fig. 3). The same result was obtained when ionic strength of the medium was increased by addition of 100 mM NMGCl or 100 mM TrisCl (not shown). Tris decreased the apparent affinity to Na, and the Na dependence curve became S-shaped. The half-saturating [NaCl] in the presence of TrisCl was 2.7 ± 0.12 mM (n = 6), and only 0.33 ± 0.02 mM (n = 6) with NMGCl in the medium. With 100 mM NMGCl, in the absence of added NaCl, 4-12% of the enzyme was phosphorylated (not shown), but it is not clear whether this phosphorylation was caused by NMG itself or by traces of Na.


Figure 3: Effect of TPP and TPB on the formation of phosphoenzyme. Bovine brain Na,K-ATPase was incubated for 10 min at 20 °C in 30 mM imidazole buffer (pH 7.4 at 0 °C), 0.1 mM MgCl(2), and varying [NaCl] and [NMGCl] (left panel) or [NaCl] and [TrisCl] (right panel), in the absence of lipophilic ions (circles) and in the presence of 300 µM TPP (squares) or 33 µM TPB (triangles). The sum of [NaCl] and [NMGCl] or [TrisCl] was 100 mM. The samples were subsequently cooled to 0 °C. 25 µM [-P]ATP (final) was added, while concentrations of other components remained unchanged. The enzyme was phosphorylated at 0 °C. After 2 s, the phosphorylation was stopped and the amount of phosphoenzyme was measured as described under ``Materials and Methods.'' The figure shows the amount of phosphoenzyme formed at 0-10 mM NaCl.



Na-dependent Dephosphorylation

Na-dependent dephosphorylation of the phosphoenzyme was examined in the absence of ADP or KCl. The experiments were performed at 10-300 mM NaCl, and the chase contained 1 mM unlabeled ATP. The Na dependence of the dephosphorylation rate coefficients, obtained by a monoexponential fit of the dephosphorylation data, is shown in Fig. 4.


Figure 4: Effect of lipophilic ions on Na-dependent dephosphorylation. Dependence of dephosphorylation rate constant on [NaCl]. Bovine brain Na,K-ATPase was phosphorylated for 60 s at 0 °C as described under ``Materials and Methods'' at varying [NaCl] in the absence of lipophilic ions (circles), with 300 µM TPP (squares) or 33 µM TPB (triangles). At zero time, a chase containing unlabeled ATP (1 mM final) was added. The dephosphorylation was stopped and the first order rate constants were estimated as described under ``Materials and Methods.''



In the absence of lipophilic ions, the dephosphorylation was stimulated by NaCl in the concentration range from 10 to 150 mM, similar to previous data by Hara and Nakao(1981) and Nørby et al.(1983). The stimulation appears to be due to a stimulation of dephosphorylation of the K-sensitive EP. It has been shown previously that the K-insensitive EP dephosphorylates directly (Nørby at al., 1983), and its transition to the K-sensitive EP is inhibited by NaCl (Klodos et al., 1994). However, because of a low relative amount of K-insensitive EP (less than 12% of the total EP, Fig. 9), neither of the two processes could significantly affect the dephosphorylation of this phosphoform. Thus, NaCl stimulation of the Na-dependent dephosphorylation was due to Na acting as a replacement, albeit poor, for K in activating the dephosphorylation of K-sensitive EP. As shown in Fig. 4, the rate coefficient of Na-dependent dephosphorylation was increased by 33 µM TPB at [NaCl] lower than 150 mM, whereas it was decreased by 300 µM TPP at all NaCl concentrations.


Figure 9: TPP effect on K-stimulated dephosphorylation of bovine brain Na,K-ATPase. The experiments were performed as in Fig. 8with bovine brain Na,K-ATPase at varying [NaCl] in the absence (circles) or in the presence of 300 µM TPP (squares). [KCl] in the chase was 20 mM (final). A biexponential function EP= EPbulletexp(-kbullett) + EPbulletexp(-kbullett) was fitted to the data like those in Fig. 8as described under ``Materials and Methods.'' The relative amount of K-insensitive phosphoenzyme, EP, the rate coefficient of the decay of EP, k, and the rate coefficient of the decay of EP, k, are shown as function of [NaCl] in panels A, B, and C, respectively.




Figure 8: [KCl] dependence of K-stimulated dephosphorylation of phosphoenzyme: Effect of TPP. The phosphorylation was performed as in Fig. 4with bovine brain (upper row) or pig kidney Na,K-ATPase (lower row) in the presence of 100 mM NaCl without (left column) or with 300 µM TPP (right column). At zero time, a K chase containing unlabeled ATP (1 mM final) and [KCl] (final), 1 mM (triangles), 20 mM (squares), or 50 mM (circles), was added. Dephosphorylation was stopped at the times shown.



ADP-dependent Dephosphorylation

Neither TPP nor TPB in concentrations up to 600 µM and 100 µM, respectively, influenced the steady state EP level at [NaCl] geq 10 mM (not shown). In a series of experiments, EP was formed in the presence of 10 to 500 mM NaCl with or without lipophilic ions for 60 s and subsequently chased with 2.5 mM ADP.

The addition of ADP in the chase produced rapid decay of the ADP-sensitive EP and exposed the slowly decaying ADP-insensitive EP (Fig. 5A). The biphasic dephosphorylation was analyzed in the following scheme


Figure 5: Effect of lipophilic ions on ADP-dependent dephosphorylation. The experiments were performed with bovine brain Na,K-ATPase as in Fig. 4at varying [NaCl] in the absence of lipophilic ions (circles) and in the presence of 300 µM TPP (squares) or 33 µM TPB (triangles). At zero time, a chase solution containing unlabeled ATP (1 mM final) and ADP (2.5 mM final) was added. Dephosphorylation was stopped at the times shown. Panel A, dephosphorylation time course at 100 mM NaCl. A biexponential function EP = EPbulletexp(-kbullett) + EPbulletexp(-kbullett) was fitted to the data like those in panel A as described under ``Materials and Methods.'' EP, the relative amount of ADP-sensitive phosphoenzyme, and k, the rate coefficient of decay in the slow phase, are shown as functions of [NaCl] in panels B and C, respectively.



where EP is a rapidly and EP is a slowly decaying phosphoenzyme form(s). EP and EP are not synonymous with the classical ADP-sensitive, K-insensitive E(1)P or the ADP-insensitive, K-sensitive E(2)P, but signify operational quantities of phosphoforms characterized by their, rapid or slow, decay. k is the rate coefficient of ADP-dependent decay, k and k are the rate constants of forward and backward transitions between the ADP-sensitive and the ADP-insensitive phosphointermediates, and k is the rate coefficient of dephosphorylation of the ADP-insensitive EP. The forward transition is accompanied by dissociation of at least 1 Na (Yoda and Yoda, 1987; Glynn, 1988; Jørgensen, 1991, 1994; Goldshleger et al., 1994). Both the backward transition, accompanied by the binding of NaCl (Post et al., 1975; Nørby et al., 1983; Nørby and Klodos, 1988), and the dephosphorylation of the ADP-insensitive phosphointermediate, reflected by the rate coefficient of the Na-dependent dephosphorylation (Fig. 4) (Klodos et al.(1981); cf. Nørby and Klodos (1988)), are dependent on [NaCl]. The rate coefficient of the decay in the slow phase is equal to the sum of k and k.

In the absence of lipophilic ions, an increase in [NaCl] resulted in: 1) an increase in the steady state amount of ADP-sensitive EP (Fig. 5, A and B), and 2) an increase in the rate of the slow decay (Fig. 5C). Both observations are in agreement with previously published data (Hara and Nakao, 1981; Nørby et al., 1983; Klodos and Nørby, 1987).

TPP caused a large decrease in both the steady state amount of ADP-sensitive EP and in the rate of the slow phase at all NaCl concentrations (Fig. 5). The opposite was seen with TPB, which caused a somewhat smaller, but significant, increase in both the steady state amount of EP and a significant increase in the slope of the slow phase (Fig. 5). In other words, the effect of TPP was similar to that of a decrease in the concentration of NaCl and the effect of TPB to an increase in [NaCl].

Similar effects of lipophilic ions were observed in experiments with Na,K-ATPase from pig kidney (Fig. 6) and shark rectal gland (not shown). Although the steady state proportion of ADP-sensitive EP measured, in the presence of 100 mM NaCl in the medium, was lower with these enzymes than with brain Na, K-ATPase (compare Fig. 5and Fig. 6at 100 mM NaCl, cf. also Klodos & Nørby(1987) and Klodos et al.(1994)), qualitative effects of lipophilic ions on both the steady state level of ADP-sensitive EP and the slope of the slow phase were the same as with the brain Na,K-ATPase.


Figure 6: ADP-stimulated dephosphorylation of pig kidney Na,K-ATPase: effect of lipophilic ions. The experiment was the same as in Fig. 5A except the source of enzyme. circles, NaCl alone; squares, +300 µM TPP; open triangles, +33 µM TPB-; or filled triangles, 100 µM TPB-.



100 µM TPB showed a more pronounced effect than 33 µM (Fig. 6). With TPP in the medium, the half-maximal effect was obtained at 30 µM (Fig. 7). In these experiments, the relative amount of the ADP-sensitive EP was estimated as the amount of phosphoenzyme removed by a 2-s ADP chase.


Figure 7: Fraction of ADP-sensitive phosphoenzyme as a function of [TPP]. The experiment was performed with bovine brain Na,K-ATPase phosphorylated for 60 s in the presence of 100 mM NaCl and varying [TPP]. An ADP chase containing unlabeled ATP (1 mM final) and ADP (2.5 mM final) was added after 60 s of phosphorylation, and the dephosphorylation was stopped after 2 s as described under ``Materials and Methods.'' The fraction of ADP-sensitive phosphoenzyme is defined here as the proportion of phosphoenzyme removed by the ADP chase of 2-s duration, DeltaEP = EP(0) - EP, and shown as percentage of EP(0).



K-dependent Dephosphorylation

TPP effect on the K-dependent dephosphorylation was tested in experiments performed with bovine brain, pig kidney, and shark rectal gland Na,K-ATPases.

The phosphoenzyme formed in the presence of 300 µM TPP showed much lower apparent affinity toward K than the enzyme phosphorylated under the same conditions but in the absence of TPP (Fig. 8). In the absence of TPP, identical dephosphorylation was observed with a K chase containing 20 or 50 mM KCl, and 1 mM KCl was almost as efficient as 20 or 50 mM KCl (Fig. 8, left column). This is clearly not the case in the presence of 300 µM TPP (Fig. 8, right column), where 1 mM KCl was far from saturating. 20 mM KCl was enough to elicit maximum dephosphorylation in the presence of lipophilic cation, and, therefore, in the experiments described below, the K chase contained 20 mM KCl.

The dephosphorylation results, similar to those in Fig. 8, were evaluated according to the following scheme

Again, EP and EP are not synonymous with the classical E(1)P or E(2)P, but signify operational quantities of different phosphoforms characterized by their decay. The rapid phase of dephosphorylation corresponds to the K-sensitive phosphoenzyme, EP, decaying with the apparent rate constant k (the rate coefficient in the presence of K), while the slow phase corresponds to the K-insensitive phosphoenzyme, EP. The rate constant of the slow decay is equal to the sum of forward transition rate coefficients, k and k.

Fig. 9shows the relative amount of the K-insensitive EP (Fig. 9A) and the apparent rate coefficient of the slow (Fig. 9B) and the rapid (Fig. 9C) phases as a function of [NaCl] without and with 300 µM TPP in the medium. The relative amount of K-insensitive EP increased with an increase in [NaCl] as expected from the previously published data (Hara and Nakao, 1981; Nørby et al., 1983; Klodos et al., 1994). The rate coefficient for the rapid phase (Fig. 9C) showed an inhibition of the rapid decay by high [NaCl]. This inhibition was not caused by a competition between NaCl and K, since even at 600 mM NaCl an increase in [KCl] from 20 to 50 mM did not affect the rapid phase of the dephosphorylation (not shown). When the phosphorylation was performed in the presence of 300 µM TPP, the rate coefficient of the rapid phase (Fig. 9C) decreased at all [NaCl] tested, while k, the rate coefficient of decay of the K-insensitive EP neither depended on [NaCl] nor did it change in the presence of TPP. Similar effects of TPP on the K-dependent dephosphorylation were observed with pig kidney (Fig. 8) and shark rectal gland Na,K-ATPase (not shown).

Is Na Essential for the Effects of Lipophilic Ions?

It has been shown previously that both the steady state ratio of the phosphoenzyme forms and their dephosphorylation kinetics are modified by high concentrations of salt (Post and Suzuki, 1991; Klodos et al., 1994). We tested, therefore, whether the effects of lipophilic ions were modified by various salts. The experiments were performed either at 100 mM [Cl], substituting most of Na by NMG, or Tris, or at 100 mM [Na], replacing most of Cl by nitrate, acetate, or sulfate (not shown). The results of the ADP and the K chase experiments are shown in Fig. 10and 11, respectively, where the experiments at 10 mM NaCl alone are shown for comparison .


Figure 10: Effect of various salts and lipophilic ions on ADP-dependent dephosphorylation. The experiments with bovine brain enzyme were performed as in Fig. 5in the presence of 10 mM NaCl alone or 10 mM NaCl with either 90 mM NMGCl, 90 mM TrisCl, or 90 mM NaNO(3), or 90 mM CH(3)COONa in the absence (empty bars) or in the presence of 300 µM TPP (light shadowed bars) or 33 µM TPB (heavily shadowed bars). The data were evaluated as in Fig. 5. Panel A, EP, the relative amount of ADP-sensitive phosphoenzyme; panel B, k, the rate coefficient of decay in the slow phase.



As seen from Fig. 10and Fig. 11, the replacement of Na by other cations or substitution of Cl by other anions caused small, but significant, changes in the relative amounts of the ADP-sensitive and of the K-insensitive phosphoenzymes. The changes were as expected from the literature, indicating that the lyotropic effects of salts on the distribution of phosphoenzymes appear already at salt concentrations as low as 100 mM.


Figure 11: Effect of various salts and lipophilic ions on Kdependent dephosphorylation. The experiments were performed as in Fig. 9and Fig. 10. The data were evaluated as in Fig. 9. Panel A, EP, the relative amount of K-insensitive phosphoenzyme; panel B, k, the rate coefficient of decay of the K-sensitive phosphoenzyme. Empty bars in the absence and shadowed bars in the presence of 300 µM TPP.



None of the substitutions affected TPP-induced decreases in the rate coefficient of the rapid decay of K-sensitive EP (Fig. 11). Moreover, the replacement of Cl for other anions did not modify the ADP-dependent dephosphorylation in the presence of lipophilic ions (Fig. 10). The only modification of the effect of the lipophilic ions on the phosphoenzyme distribution and the ADP-dephosphorylation kinetics was seen when Na was replaced by other cations. Although the relative amount of ADP-sensitive EP in the presence of 10 mM NaCl and 90 mM NMGCl was significantly higher than with 10 mM NaCl alone, the effect of lipophilic ions on the phosphoenzyme distribution remained the same as with 10 mM NaCl without NMGCl. The lipophilic ions effect on the distribution of phosphoenzymes disappeared completely in the presence of Tris (Fig. 10A). The rate coefficient of the decay of the ADP-insensitive EP was decreased by both Tris and NMG in the absence of lipophilic ions, but returned to a value characteristic for 10 mM NaCl in the presence of TPB (Fig. 10B). TPP, however, did not decrease the coefficient to the same value as with 10 mM NaCl + TPP but to a value measured with lipophilic cation at 100 mM NaCl. At present, we have no explanation of this difference in NMG or Tris modification of the effect of TPP on the distribution of phosphoenzymes on one hand, and on the rate coefficient for the slow phase of the ADP-dependent dephosphorylation on the other.

Phosphoenzyme as Function of Time at Low [NaCl]

We observed that although the two lipophilic ions did not affect the steady state EP level at [NaCl] higher than 10 mM, they both showed a pronounced effect on the steady state level at lower [NaCl]. The last observation seemed to be in conflict with the results of 2-s phosphorylation (Fig. 3). To elucidate this apparent controversy, we measured the time course of EP formation at 1, 3, and 10 mM NaCl and 33 µM TPB or 300 µM TPP. The results are shown in Fig. 12.


Figure 12: Time course of phosphorylation at three different [NaCl]. The experiments were performed as described under ``Materials and Methods'' with bovine brain Na,K-ATPase and in the presence of 1 mM (left panel), 3 mM (middle panel), or 10 mM (right panel) NaCl alone (circles); + 300 µM TPP (squares) or 33 µM TPB (triangles). 100% corresponds to the steady state value obtained at 100 mM NaCl.



In accordance with Fig. 3, the phosphorylation observed at 2 s was only slightly, if at all, influenced by the lipophilic ions. The experiments performed in the absence of lipophilic ions (Fig. 12) showed, however, that: 1) in the presence of low NaCl (1 and 3 mM) an initial high formation of EP was followed by a decrease in EP to a steady state level, which was reached after more than 1 min (not shown), 2) the size of the ``overshoot,'' i.e. the difference between the 2-s value and the steady state level of EP, decreased with increasing [NaCl], and, at 10 mM NaCl, no overshoot was observed, and 3) TPB increased the overshoot by decreasing the steady state levels at 1 and 3 mM NaCl. In contrast, 4) in the presence of 300 µM TPP, no overshoot was observed at any [NaCl].

ATPase Activity

The rate of ATP hydrolysis in the absence of K at 3 mM ATP, 3 mM MgCl(2) increased with [NaCl] up to about 400 mM, whereas inhibition was caused by higher NaCl concentrations (Fig. 13). A concentration-dependent shift of the curve to the right was observed with TPP (2 and 5 µM), i.e. higher concentrations of Na were needed for activation as well as for inhibition. TPB (5 and 10 µM) had the opposite effect. (The concentrations of lipophilic ions in these experiments were decreased because of the very low protein concentrations used.)


Figure 13: Na-ATPase activity as a function of [NaCl]. The experiments were performed with bovine brain Na,K-ATPase as described under ``Materials and Methods.'' Empty circles, activity in the presence of NaCl. Panel A, effect of 5 (empty triangles) or 10 (filled triangles) µM TPB on the activity. Panel B, effect of 2 (empty squares) or 5 (filled squares) µM TPP.



When the measurements were repeated in the presence of 20 mM KCl, the maximal hydrolysis rate was increased by a factor of 25 and was obtained at [NaCl] 150 mM (Fig. 14). The effect of lowering [KCl] was to decrease the maximal hydrolysis rate and to shift the curve to the left, i.e. the maximal hydrolysis rate was obtained at a lower [NaCl] (Fig. 14A). The effect of 20 µM TPP was equivalent to decreasing the concentration of K (Fig. 14B).


Figure 14: Na,K-ATPase activity. The experiments were performed with bovine brain Na,K-ATPase as described under ``Materials and Methods.'' Panel A, effect of K on the activity. The activity was measured as a function of [NaCl] in the absence of KCl (empty circles) or with 2.5 mM KCl (filled circles), 5 mM KCl (empty squares), or 20 mM KCl (filled squares). Panel B, effect of TPP on the activity in the presence of 5 mM KCl. The activity in the absence of TPP, empty squares, and with 20 µM TPP, empty circles.



pNPPase Activity

As shown in Fig. 8and Fig. 9, TPP affected very strongly the properties of the K-sensitive EP. To further elucidate the interaction of the enzyme with K, we measured the activity of K-stimulated pNPPase, which is assumed to reflect potassium-sensitive steps in the Na,K-ATPase reaction mechanism (cf. review by Glynn(1985)). The activity was measured as a function of [KCl] in two series of experiments. The first set was performed with varying [KCl], and the ionic strength was kept constant with choline or NMG chloride. Under these conditions, TPP had very little, if any, effect on the activity (not shown).

In the second set of experiments (Fig. 15), both NaCl and KCl were present, and the sum of [NaCl] and [KCl] was kept equal to 150 mM. The experiments were carried out without or with 100 µM ATP. The effect of TPP was clearly seen both in the presence and in the absence of ATP (Fig. 15). With ATP in the medium, TPP caused both a decrease in the apparent affinity for KCl and a change in the shape of the K activation curve.


Figure 15: Effect of K and 100 µM ATP on pNPPase activity in the presence of both Na and K. The experiments were performed at 37 °C with bovine brain Na,K-ATPase as described under ``Materials and Methods.'' The medium contained 20 mM MgCl(2), 10 mM pNPP, 30 mM imidazole buffer, pH 7.5, at 20 °C, KCl and NaCl. The sum of [NaCl] and [KCl] was equal to 150 mM. The experiments were performed in the absence (left column) or in the presence (right column) of 100 µM ATP. Squares, activity in the presence of 300 µM TPP. Lower row, expanded abscissa.



Is the Effect of Lipophilic Ions Caused by Their Charge?

Our original assumption that the effects of lipophilic ions were caused by their charge required an experimental proof. To rule out the possibility that the observed effects were due to an irreversible protein modification by lipophilic ions, we examined whether the effect of TPP would disappear after removal of the membrane-bound TPP. In these experiments, the enzyme was incubated with 100 or 300 µM TPP and subsequently repeatedly washed by centrifugation and resuspension in TPP-free medium. As a control, TPP-bound enzyme was incubated and washed in a medium containing TPP, and TPP-free enzyme was exposed to the same treatment but in the absence of TPP. After the subsequent phosphorylation, the phosphoenzymes were probed with a 2-s 2.5 mM ADP chase or a 2-s 1 mM K chase, the concentration of KCl at which the TPP modification of the phosphoenzyme behavior was most apparent (Fig. 8). The enzyme, pre-exposed to TPP and subsequently washed in TPP-free medium, and the TPP-free enzyme formed identical phosphointermediates with respect to their K and ADP sensitivity (not shown), thus excluding an irreversible modification of the protein by TPP.

In another series of experiments, we investigated whether the effect of TPP was reversed by a subsequent addition of TPB and vice versa (Fig. 16). In these experiments, the enzyme was incubated in the phosphorylation medium containing 100 mM NaCl and 50 or 300 µM TPP. After 15 min, 3.3 or 33 µM TPB was added, and the incubation was allowed to proceed for another 15 min (Fig. 16, left panel). In a parallel series of experiments, TPB was present during the first 15 min of incubation and TPP was included during the next 15 min (Fig. 16, right panel). The enzymes were then phosphorylated and the phosphoenzymes were probed with a 2-s ADP chase. It is obvious from Fig. 16that the effect of one lipophilic ion was reversed by a lipophilic ion of the opposite charge. The phenomenon was concentration-dependent but was independent of the sequence of additions, i.e. of the charge of lipophilic ion that was first ``seen'' by the enzyme. Thus, our conclusion from these experiments is that the effect of lipophilic ions on the properties of the enzyme is related to their electric charge.


Figure 16: Effect of sequential incubations with TPP and TPB on ADP sensitivity of phosphoenzyme. Left panel, bovine brain Na,K-ATPase was incubated at 20 °C in a medium containing 100 mM NaCl, 0.1 mM MgCl(2), and 30 mM imidazole buffer in the presence of 50 or 300 µM TPP for 15 min. Subsequently, 0, 3.3, or 33 µM TPB was added for an additional 15 min. Right panel, the enzyme was incubated first for 15 min with 3.3 or 33 µM TPB, then 0, 50, or 300 µM TPP was added for the following 15 min. After the incubation, the samples were cooled to 0 °C and phosphorylated for 60 s at 100 mM NaCl, 0.1 mM MgCl(2), and 25 µM [-P]ATP. The ADP sensitivity was probed as in Fig. 7, and the fraction of phosphoenzyme remaining after a 2-s chase is shown. The same experiment performed in the absence of lipophilic ions is shown for comparison (labeled ``100 mM NaCl'').




DISCUSSION

The first question to consider is whether the lipophilic ion effect on the Na,K-ATPase reaction is due to a modification of the intramembrane electric field. The experiments with sequential additions of lipophilic ions to the medium (Fig. 16) indicate that the effects of TPP and TPB are caused by their charge. We ascribe therefore the effects of lipophilic ions to modifications of the electric field in the membrane.

In the present study, our attention was focused on the reaction steps where Na and K are known to bind to or leave the enzyme. To discriminate between Na and K interactions with intra- and with extracellular sites, we studied partial reactions involving dephosphoforms, known to have their inward-facing sites open, and phosphoenzymes, where the outward-facing sites are accessible (cf. reviews by Glynn and Karlish(1990), Robinson and Pratap(1993), and Vasilets and Schwarz(1993)). An attempt was also made to correlate the induced changes in the partial reactions with the effects on the overall reaction cycle.

K and Na Interaction with Intracellular Sites

Na and K binding to Na,K-ATPase and transitions between dephosphoenzymes, E(2)(K(n)) and E(1)Na(m), were investigated using eosin as an indicator of conformations (Skou and Esmann, 1983). As documented under ``Results,'' neither affinity toward Na nor K were affected by the lipophilic ions implying that the binding of Na or K to the E(1) form of the enzyme and the occlusion of intracellular K are not influenced by the intramembrane electric field. Moreover, the formation of phosphoenzyme, which is a function of Na binding to the intracellular sites, was not affected by the lipophilic ions. Thus, the interaction of Na,K-ATPase with the intracellular Na and K appears to be independent of the intramembrane electric field. This conclusion is in agreement with the results obtained with Na,K-ATPase reconstituted into lipid vesicles (Rephaeli et al., 1986a).

Na and K Interaction with Extracellular Sites

Phosphorylation of Na,K-ATPase from ATP leads to a closure of inward-facing cation binding sites and to an opening of outward-facing cation binding and release sites (cf. reviews by Glynn and Karlish(1990) and Robinson and Pratap(1993)). Na release to the external medium accompanies the transition of ADP-sensitive to potassium-sensitive phosphointermediates (Yoda and Yoda, 1987; Glynn, 1988; Jørgensen, 1991, 1994; Goldshleger et al., 1994). The effect of lipophilic ions on the composition of the phosphoenzyme pool, the (spontaneous) Na-dependent dephosphorylation, and the reactivity of phosphoenzymes toward K and ADP in the K and ADP chase experiments will thus reveal modifications in the properties of extracellular cation binding sites.

Steady State Fraction of ADP- or K-sensitive Phosphoenzymes

Lipophilic ions affected the steady state composition of the EP pool, but they affected neither the formation of EP nor, at NaCl concentrations higher than 3 mM, the steady state level of EP. The ratio between the ADP-sensitive and the ADP-insensitive EP is determined by the dissociation of Na or salt from the ADP-sensitive EP and the rebinding of Na or salt to the ADP-insensitive phosphoform (see Klodos et al., 1994). The steady state ratio was decreased by TPP (Fig. 5, Fig. 6, and Fig. 7) and increased by TPB ( Fig. 5and Fig. 6) and, although the EP ratio was affected by various salts, the modification of the ratio by lipophilic ions required the presence of Na (Fig. 10).

Despite a significant TPP-induced decrease in the fraction of ADP-sensitive EP, the proportion of K-insensitive EP seemed to be only slightly decreased by TPP ( Fig. 8and Fig. 9). However, the relative amounts of the phosphoenzymes disappearing in the two phases of dephosphorylation correspond to the initial, steady state amounts of these phosphoforms only when the rate of dephosphorylation for one of the intermediates is much greater than the rate of transition between phosphoforms (k, k, and k in ) (Klodos et al., 1981). Thus, it is to be expected that the experimentally determined amount of K-insensitive EP is larger in the presence of TPP, since this compound decreases the rate coefficient for the rapid decay of K-sensitive phosphoform, but leaves the transition rate coefficients unaffected (see Fig. 9, B and C) This expectation is supported by the fact that even in the absence of TPP the fraction of K-insensitive EP seemed to increase when the rate coefficient of the rapid phase was decreased by lowering [KCl] in the chase (to 1 mM instead of 20-50 mM, Fig. 8).

Kinetics of Na- or ADP-dependent Dephosphorylation

The effect of lipophilic ions on the steady state ratio of the ADP-sensitive to the ADP-insensitive EP and the specific requirement for Na for this effect leads to a question whether the effect was caused by a modification of the release of Na from the extracellular site or by changed rebinding of Na to extracellular sites on the ADP-insensitive EP. The present data allow us to analyze only the modification of Na rebinding to the ADP-insensitive EP, by examining the effect of lipophilic ions on the kinetics of Na- and ADP-dependent dephosphorylation.

Under conditions where the K-sensitive EP amounted to 86-90% of the EP level, the rate coefficient of Na-dephosphorylation reflects only the forward dephosphorylation of this EP form, k in (see also Klodos et al.(1981)). In the ADP chase experiments the rate coefficient of the slow phase reflects both forward dephosphorylation and backward transition of the EP, k and k in .

Lipophilic ions affected the rate coefficient for both the Na-dependent dephosphorylation and the slow decay of ADP-insensitive EP. TPP caused a decrease in both the rate coefficient for Na-dependent dephosphorylation (Fig. 4) and of the slow decay (Fig. 5), while TPB induced an increase in both coefficients. TPB induced a shift of the Na dependence curve for the Na-dependent dephosphorylation to lower [NaCl] (Fig. 4), but did not change the maximal value of the rate coefficient. At 100 mM NaCl, TPB increased the rate coefficient of the slow decay of the ADP-insensitive EP (Fig. 5), whereas the rate coefficient of the Na-dependent dephosphorylation remained virtually unchanged (Fig. 4). This could indicate that the TPB-dependent acceleration of the slow decay is caused by an increase in affinity for Na for the backward transition of EP to EP. It is, however, not clear whether the change in the backward transition is sufficient to cause the observed shift in the ratio of the ADP-sensitive to the ADP-insensitive EP.

Kinetics of K-dependent Dephosphorylation

At saturating [KCl], TPP elicited a decrease in the rate coefficient of the rapid decay, i.e. the dephosphorylation of the K-sensitive EP ( Fig. 8and Fig. 9), and the apparent affinity for K (Fig. 8).

To evaluate whether this observation entailed a TPP-induced increase in the dissociation constant, K, of K-bound phosphoenzyme, E(2)PK, the results of experiments in Fig. 8were analyzed according to the scheme

The reaction sequence does not include K-insensitive EP comprising, at 100 mM NaCl, less than 10% of the phosphoenzyme.

When k is much lower than both k and k, the rate coefficient for the rapid decay k = k/(1 + K/[K]), K being the dissociation constant for K, k/k. It is obvious from the equation that the ratio of k at constant nonsaturating [K], i.e. 1 mM KCl, to k, both determined in the absence or in the presence of lipophilic cation, should be the same unless K is affected by TPP.

The rate coefficient for the rapid decay, k, was a saturable function of [KCl] both in the absence and in the presence of TPP, i.e.k values at 20 mM KCl were identical with those at 50 mM KCl (Table 1). We assumed therefore that they represent k. k obtained at 100 mM NaCl in the absence of lipophilic cation were virtually the same, 1.9 and 2.4 s, for the bovine brain and the pig kidney Na,K-ATPase, respectively. The corresponding values in the presence of 300 µM TPP were 0.9 s for both enzymes. In the presence of 1 mM KCl in the chase, the k/k ratio was about 0.8 for both brain and pig kidney enzyme and decreased to 0.5 in the presence of TPP (Table 1), implying about a 4-fold increase in K in the presence of lipophilic cation.



The conclusion about the effect of lipophilic cation on the reactivity of the K-sensitive EP toward the extracellular cations is supported by a similar evaluation of the spontaneous, Na-dependent, dephosphorylation. TPP decreased significantly the rate coefficient for Na-dependent dephosphorylation and, less conclusively, it seemed to decrease the apparent affinity for extracellular Na (Fig. 4). TPB on the other hand moved the curve to lower [NaCl] (Fig. 4), thus indicating an increased apparent affinity for Na binding to the K-sensitive EP's extracellular cation binding site.

Thus, based on the analysis of both the Na-dependent dephosphorylation and the ADP or K chase experiments, the intramembrane charge alters: 1) Na binding to the ADP-insensitive EP affecting the backward transition to the ADP-sensitive EP, 2) K-sensitive EP's affinity toward K or Na, and 3) the properties of the K-sensitive EP-cation complex, reflected in the dephosphorylation rate constant. This suggests that the intramembrane electric field affects Na and K passage to and from the outward-facing cation binding pocket and alters kinetic properties of EP-cation complexes. A change in the kinetic properties of the K-sensitive EP-cation complex could reflect a difference in the way the cation was bound and might also result in changed properties in the subsequently formed E(2)-cation complex. This appears to be the case, since TPP seems to prevent occlusion of extracellular cations.

Occlusion of Extracellular Na and K

Initial formation of phosphoenzyme in the presence of 1-3 mM NaCl was followed by a decrease in the amount of phosphointermediate, so-called overshoot (Fig. 12). A generally accepted explanation of such an overshoot is a decrease of the phosphoenzyme formation due to an accumulation of E(2)(cation) form. The overshoot was previously observed with K (Mårdh, 1975; Klodos and Nørby, 1979), but also in the presence of Na alone, when low ionic strength and low [ATP] did not support the transition of E(2)(Na) to the E(1) form (Klodos and Ottolenghi, 1985). In the latter case, Na acted as K congener stimulating the dephosphorylation of the K-sensitive EP. The transition of E(2)(cation) into the phosphorylatable E(1) form requires either high [ATP] or, at low [ATP], high [NaCl], or both, and, in our experiments, with 25 µM ATP in the medium, 10 mM NaCl was sufficient to prevent the accumulation of the occluded form (Fig. 12).

The overshoot observed in the absence of K was increased by TPB, while in the presence of TPP there was no overshoot. Since the phosphorylation was not affected by lipophilic cation, the lack of overshoot in the presence of TPP could be due to either an increased deocclusion of the E(2)-cation complex or an inhibition of formation of the E(2)(Na) or both. As lipophilic cation did not affect transitions between the dephosphoforms, the most likely explanation is that TPP prevents formation of E(2)(Na). The opposite effect of TPB on the overshoot indicates that lipophilic anion increased the occlusion of Na.

ATPase Activity

Effect of lipophilic ions on the interaction of Na and K with the extracellular sites was also seen in the hydrolytic activity of Na- and Na,K-ATPase. The bell-shaped Na dependence curve of Na-ATPase activity might be interpreted as an expression of a shift in the rate-limiting step from the dephosphorylation of E(2)P, accelerated by [NaCl] leq 400 mM, to the E(1)P &rlhar2; E(2)P transition, inhibited by high [NaCl] acting at a low affinity extracellular binding site (Glynn and Karlish, 1976; Kaplan and Hollis, 1980; Kaplan, 1982). It is in agreement with this interpretation that 1) with TPP, which slowed down the dephosphorylation of E(2)P and increased the rate of transition, the Na dependence curve was moved to higher [NaCl], i.e. the maximal hydrolytic activity was obtained at significantly higher [NaCl] and a much smaller inhibition was observed at very high [NaCl], and 2) TPB, accelerating both the dephosphorylation of E(2)P and its backward transition, decreased the concentration of NaCl required for the maximal activation of hydrolysis and increased the degree of inhibition at high [NaCl] (Fig. 13).

At saturating substrate concentrations, the effect of K is to accelerate dephosphorylation and ATP hydrolysis through binding to the extracellular K site, maximal activity being obtained at 20 mM KCl and 150 mM NaCl. Decreasing under these conditions, [K] below 20 mM, decreased the hydrolysis rate and the Na concentration at which maximal hydrolysis rate was obtained (Fig. 14A). The effect of TPP was equivalent to decreasing the K concentration (Fig. 14B), consistent with a decreased affinity for K, as demonstrated above.

pNPPase Activity

Lack of effect of TPP on pNPPase activity in the presence of K suggests that the hydrolysis of pNPP does not involve interaction of K with the extracellular sites. This observation is in the agreement with the analysis of the kinetics of K-dependent pNPPase by Beauge and Berberian(1983) and Berberian and Beauge(1985), who pointed out that ``during phosphatase activity . . . , the most abundant form is a nonoccluded E(2) and that at least one of the mechanisms of potassium stimulation of that activity is to take the enzyme into the E(2) state'' (Berberian and Beauge, 1985).

At low concentrations of KCl, the activity was strongly inhibited by NaCl (not shown), but the inhibition was almost completely reversed by low [ATP] (Fig. 15). As previously suggested by Post et al.(1972) and by Drapeau and Blostein(1980), this activating effect of ATP might be caused by an increase in the concentration of E(2) due to the K-stimulated dephosphorylation of E(2)P formed from ATP through the ``physiological route'' (cf. Glynn, 1985). The fact that under these conditions TPP affected the pNPPase activity indicates an involvement of the extracellular potassium-dependent steps of the reaction and supports this hypothesis. More surprising is that TPP also affected the activity in the absence of ATP when both NaCl and KCl were present (Fig. 15). Since the transition between E(2)K and E(1)Na was not affected by the lipophilic cation, this implies that also in this case the reaction passes through some intermediates with their outward-facing sites open.

Electric Field in the Membrane and Cation Interactions with Na,K-ATPase: Conclusions

The pronounced effect of lipophilic ions (Läuger, 1991a) and of the transmembrane potential on Na and K interaction with the outward-facing sites (Gadsby et al., 1991; Rakowski, 1991; Rakowski et al., 1991; Schwarz and Vasilets, 1991; Vasilets et al., 1991; Gadsby et al., 1992; Stimers et al., 1993; Vasilets and Schwarz, 1993; Hilgemann, 1994; Sagar and Rakowski, 1994) was taken as evidence that ``extracellular Na and K ions have to pass through a narrow access channel, which traverses a substantial fraction of the membrane field, before they can interact with their binding sites on the Na,K-ATPase molecule'' (Gadsby et al., 1992). The previously observed weak or even lacking membrane potential dependence of Na and K interaction with the inward-facing sites (Rephaeli et al., 1986a; Goldshlegger et al., 1987) and several other observations led to the conclusion that these sites are formed as shallow, low field, wells (Goldshlegger et al., 1987; Apell, 1989; Läuger, 1991, a and b).

Our results are in accord with this hypothesis. Lipophilic ions affect the interaction of Na and K with the extracellular sites, immersed in the membrane and thus influenced by the intramembrane electric field, but do not influence the interaction with the intracellular sites. The electric field-dependent modification of the interaction of Na,K-ATPase with the extracellular cations was revealed both in the backward transition of the ADP-insensitive to the ADP-sensitive EP and in the Na and K reactivity toward and the kinetic properties of the K-sensitive EP. The effect of lipophilic ions on the transition between the phosphoforms is in agreement with the data of Rephaeli et al. (1986b), who showed a direct influence of the transmembrane potential on the rate of transition. Our results show that the rebinding of Na to the ADP-insensitive EP is affected by the intramembrane electric field, but they do not exclude that the Na deocclusion and/or release could also be affected. The data indicate that the intramembrane electric field alters the affinity for extracellular K, supporting the notion that the electric field modifies K binding to the outward-facing sites (Rakowski, 1991; Rakowski et al., 1991; Schwarz and Vasilets, 1991; Stürmer et al., 1991; Vasilets et al., 1991; Stimers et al., 1993; Vasilets and Schwarz, 1993; Sagar and Rakowski, 1994). Moreover, since the dephosphorylation rate constant and the ability to occlude extracellular cations also appear to be altered, our results suggest some modification of the phosphoenzyme-cation complex itself by the electric field.

But could the effect of TPP or TPB be caused 1) by lipophilic ion-dependent changes in surface potential which in turn could influence the access of cations to their extracellular binding/release sites or 2) by some charge-dependent changes in protein structure? If the first were true, a significant modification of lipophilic ion effects by an increasing ionic strength of the medium, i.e. by ``screening'' of the surface charge, should be expected. We observed some modification of the interaction of the enzyme with intracellular Na in measurements of the formation of EP. The effect of the lipophilic ions on the formation of EP which we reported previously (Klodos and Plesner, 1992) disappeared when the ionic strength of the medium was increased. However, neither the interaction with extracellular Na nor the interaction with extracellular K appeared to be affected. The first was shown in ADP chase experiments performed in the presence of 10 mM NaCl without or with 90 mM NMGCl, and the latter in K chase experiments, where an increase in KCl concentration from 20 to 50 mM in the K chase i.e. increase in the total salt concentration in the medium from 120 to 150 mM, did not affect the K-dephosphorylation. Thus, the lipophilic ion effect of the cation interaction with the extracellular sites cannot be ascribed to changes in the surface charge.

Interaction between the introduced charge in the membrane and charged residues in the protein near or inside the cation binding sites could result in some modification of the protein structure. In this case, one would expect that lipophilic cation and anion would modify different charged residues. The fact that TPP and TPB have opposite effects speaks against this explanation since it is difficult to imagine that a modification of different residues would elicit exactly opposite effects on cation accessibility. However, the fact that both the dephosphorylation rate constant of the K-sensitive EP and its ability to occlude extracellular cations appear to be altered indicates some modification of the EP-cation complex.


FOOTNOTES

*
This work was supported in part by grants from The Danish Medical Research Council and by The Biomembrane Research Center, University of Aarhus. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Institute of Biophysics, University of Aarhus, Ole Worms Allé 185, DK-8000 Aarhus C, Denmark. Tel.: 45-8942-2937; Fax: 45-8612-9599; ik{at}mil.aau.dk.

Recipient of a stipend from the Danish Science Research Council. Permanent address: Dept. of Biochemistry, Moscow State University, Russia.

(^1)
The abbreviations used are: TPP, tetraphenylphosphonium; TPB, tetraphenylboron; Na,K-ATPase, (Na + K)-stimulated adenosine triphosphatase; K-pNPPase, K-stimulated p-nitrophenylphosphatase; E(1), Na,K-ATPase form with high affinity toward ATP and Na; E(2), Na,K-ATPase form with high affinity toward K and low affinity to ATP; EP, phosphoenzyme; E(1)P(Na), phosphoenzyme with m occluded Na ions, resistant to K and sensitive to ADP; EP, ADP-sensitive phosphoenzyme; EP, phosphoenzyme insensitive to ADP; E(2)P, phosphoenzyme sensitive to K; EP, phosphoenzyme sensitive to K; EP, K-insensitive phosphoenzyme; E(2)P(K), phosphoenzyme with n occluded K ions; NMG, N-methyl-D-glucamine; MES, 4-morpholineethanesulfonic acid.


ACKNOWLEDGEMENTS

Inge Raungaard and Angielina Tepper are gratefully acknowledged for their excellent technical assistance. We thank H.-J. Apell for the gift of lipophilic ions and Mikael Esmann for his help and advice in carrying out the experiments with eosin and for helpful discussions. We thank Sergey N. Fedosov, Jens G. Nørby, Robert L. Post, and Igor W. Plesner for many stimulating discussions, encouragement, and help in writing the manuscript.


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