Na,K-ATPase characterized in artificial membranes. 2. Successive measurement of ATP-driven...

Molecular Membrane Biology, 1994, 11, 247-254

Na,K-ATPase characterized in artificial membranes. 2. Successivemeasurement of ATP-driven Rb-accumulation, ouabain-blocked Rb-fluxand palytoxin-induced Rb-efflux

B. M. Aimer and M. Moosmayer

Laboratory of Expérimental Cell Therapeutics,Geneva University Médical School,CH-1211 Geneva 4, Switzerland

Summary

The Na,K-ATPase is a multifunctional System anchored in thémembrane of eukaryotic cells; it is responsible for théestablishment and régulation of thé Na/K balance of cell andorganism by a stoichiometric mechanism linking Na extrusionto K uptake and ATP hydrolysis. The receptor for cardioactivesteroids such as digoxin and ouabain is located at théextracellular surface of thé System. Conversely, palytoxin, thémost potent animal toxin, exerts its toxic effect by creating non-specific leaks in thé cell membrane leading to K-efflux and influxof Na and Ça ions. Ouabain prevents thé pore-forming action ofpalytoxin in cells and therefore Na,K-ATPase is suspected to bethé common receptor of ouabain and palytoxin. We hâvedeveloped an artificial membrane System to déterminestructure functJon relationships and ligand interactions ofpurified Na,K-ATPase: two-sided, bi-directional ATP-filledliposomes. In this System, ATP-driven eeRb accumulation, arrestof 86Rb-uptake by ouabain, and palytoxin-induced 86Rb-leakwere measured successively in thé same préparation. Ouabainprevented thé leak when thé enzyme was ouabain-sensitive(rabbit kidney) but not when it was ouabain-resistant (rat kidney).On thé basis of thèse data in conjunction with conformationalanalyses, allosteric conformational compétition for thé ouabain-palytoxin antagonism is proposed.

Keywords: ATP-filled liposomes, Rb-accumulation, ouabain-blocked Rb-flux, palytoxin-induced Rb-leak, ouabain antagonism.

Abbreviations: NKA, NaK-ATPase, PYX, palytoxin; OUAB,ouabain; B, borate.

Introduction

The Na,K-ATPase (NKA; EC 3.6.1.37) is an ubiquitousmembrane transport System composed by two trans-membrane subunits, namely an a polypeptide of 110 kDa anda /3 glycoprotein of 50-60 kDa (Sweadner 1989, Lingrel et al.1990). It is a multifunctional System with thé following activitiesamongst others: (i) ATPase activity, (ii) electrogenic Na-Kantiport, and (in) receptor for cardioactive steroids {Hansen1984, Goto et al. 1992, Repke and Schôn 1992, Skou andEsmann 1992). The cardioactive steroids are considered tobe thé only compounds interacting with NKA specifically(Blaustein 1993, Schoner 1993).

However, in 1981 Habermann étal, and Ishida étal.proposed that thé NKA System may serve also as receptorfor palytoxin since ouabain was found to protect cells fromthé toxin. Palytoxin is thé most potent animal toxin (Mooreand Scheuer 1971) and thé largest and most complicatednon-protein organic molécule ever found in nature {Schimizu1983); it is excreted by corals for defence against fish. It forms

iTo whom correspondence should be addressed.

pores for Na and K ions across thé cell membrane, théprésence of NKA being a prerequisite for this effect(Habermann nd Chatwal 1982). That ouabain preventedpalytoxin action via NKA and not by an unspecifiedmechanism was demonstrated in erythrocytes isolated fromthé rat, a poorly ouabain-sensitive animal, in which thépalytoxin-induced pore formation was prevented weakly byouabain (Habermann 1989). It was also shown that thé sugar,rather than thé steroid, part of thé cardioactive steroidsantagonized thé palytoxin action indicating that thé palytoxinreceptor and thé ouabain receptor were overlapping but notidentical (Ozaki étal. 1984). Taken togehter, ail théexpérimental évidence suggests that palytoxin might form apore through thé NKA molécule (Habermann 1989). Yet théconcept of NKA being thé palytoxin receptor has not yet beenaccepted generally since Na-sensitive (Sauviat et al. 1987)or Ca-permeable channels (Muramatsu et al. 1988) and théNa/H exchange System (Frelin et al. 1990, Yoshizumi et al.1991) hâve also been proposed as primary targets.

Experiments in proteoliposomes containing a préparationof NKA of undefined source showed a slightly increaseduptake of 22Na ions by thé liposomes when palytoxin wasadded to thé suspension (Yoda et al. 1991). Uptake of 86Rbion by thé same préparations was decreased by -50%.However, thé transmembrane 22Na flux seemed to besensitive to externally added ouabain which would, therefore,not correspond to transport activity of right-side-out orientedpumps which should accumulate 86Rb ions in an ouabain-sensitive manner only. The ouabain-sensitivity of thé 86Rbuptake was not shown, thus thé flux could correspond todiffusion. Furthermore, thé palytoxin-induced pore shouldlead rather to an overall decrease of thé internai isotopecontent and not to an increase since thé ions are expectedto leave thé vesicles through thé channel during thé washingprocédure in isotope-free médium (Yoda et al. 1991).

To clarify thé still unclear aspects of NKA-palytoxininteraction, a new model System was used in thé présentstudy: two-sided bi-directional liposomes, in which half of théNKA molécules are right-side out and thé other half inside-out and in which both pump populations can be activated andinhibited successively by sided and timed 86Rb, ATP,ouabain and digoxin addition (Rey et al. 1987, Anner et al.1990). The use of this System, in which thé side of action aswell as thé potency of a pump inhibitor can be determinedsimultaneously, has contributed to thé characterization of anendogenous inhibitor with physicochemical properties distinctfrom ouabain: isomeric ouabain (Anner and Haupert 1993,Tymiak étal. 1993).

In this model System ouabain-sensitive 86Rb accumulationby thé ATP-filled liposomes was induced first; to verify thatail transmembrane 86Rb uptake was mediated by thé NKAsystem, ouabain was added: ail 86Rb-uptake was arrestedwithout leak. When palytoxin was added during 86Rb-accumulation, thé liposomes lost isotope during thé washing

248 S. M. Anner and M. Moosmayer

process in a palytoxin dose- and time-dependent manner,demonstrating that a leak had been created in theirmembrane. No effect was seen in thé absence of pumpprotein. When 86Rb accumulation had been stopped withouabain first and palytoxin was added later, thé pore-formingactivity of palytoxin was greatly attenuated. An allosteric toxininteraction model is proposed to account for thé data inconjunction with thé preceding conformational analyses(Anner et al. 1994).

Results

Successive measurement of ATP-driven Rb-accumulation,ouabain-blocked Ftb-ftux and palytoxin-induced Rb-efflux

The putative pore-forming efffect of palytoxin on thé isolatedNKA was tested in thé ATP-containing two-sided liposomemodel in which thé activity of thé pumps in cellular orientationcan be measured, a prerequisite for testing palytoxin, a largehydrophilic molécule with a molecular weight of 2678-5,known to interact with thé extracellular surface of thé NKAmolécule (Habermann 1989). The concentration for half-maximal activation of Rb-uptake is ~100u,M (Rey et al.1987). However, a concentration of 10 U.M 86RbCI wasselected to lower thé transport rate and to obtain linear initialvelocities for several minutes with negligible changes of théinternai Na, Rb and ATP concentrations. Upon addition of10 U.M external ̂ Rb, thé activity of thé right-side-out orientedpumps led to 86Rb accumulation (Figure 1A); palytoxin(PYX), added to liposomes which had taken up 86Rb for2 min, provoked thé release of thé entire internai 86Rb(Figure 1B); thus, palytoxin formed a pore in liposomescontaining reconstitued NKA molécules and ail entrapped86Rb leaked out during thé gel filtration step used to removethé 86Rb not associated with thé liposomes. By contrast,when ouabain was added (alone), thé active 86Rb-uptakewas arrested, no pore was formed, and thé liposomesremained perfectly tight (Figure 1C). The tightness of théE2-ouabain complex to 86Rb ions in this situation is inagreement with thé blockage by ouabain of a K-permeabilitymediated by thé E2-form (Anner et al. 1994). When ouabainwas added prior to palytoxin, thé pore formation was greatlyreduced and only -20% of thé internai 86Rb leaked out ofthé liposomes (Figure 1D). Thus, palytoxin formed a pore bya ouabain-sensitive mechanism. When 10 U.M palytoxin wasadded to liposomes prepared without NKA, no pore formationwas observed for 2 min (data not shown).

Borate enhances pore formation by palytoxin

In cells, borate has been shown to enhance thé potency ofpalytoxin (Habermann 1989); similarly, borate enhances théinteraction of isolated NKA with palytoxin (Grell et al. 1988).To see whether this effect could be reproduced with purifiedNKA in artificial membranes, borate (B) was added first inthé absence of palytoxin to rule out an effect on thé active86Rb-transport or on thé passive liposome permeability(Figure 2A). In thé présence of 1 nM palytoxin, no poreformation was seen without borate (Figure 2B). By contrast,when thé concentration of palytoxin was increased 10-fold,

-ÛCC

C

ai û

minFigure 1. Sequential measurement of active, ouabain-inhibition of86Rb-uptake and ouabain-sensitive palytoxin-induced leak. NKA-liposomes filled with 50 mM ATP and 220 mw Na were incubated at25°C in thé présence of 5 JIM external 86Rb and thé isotopeaccumulation by thé right-side out oriented (cellular orientation) NKApopulation (•. O) measured in 5 (il aliquots as described inExpérimental procédures for 8 min in thé absence of toxins (A). When10 HM palytoxin was added (•), ail internai 86Rb leaked out duringthé gel filtration step used for removal of external isotope (B). When100 JIM ouabain was added at 2 min, thé 86Rb-uptake was blockedand ail internai 86Rb remained entrapped (C). When 100 HM ouabainwas added at 2 min and then 10 |IM palytoxin at 3 min, palytoxin nolonger formed a pore (D). Each experiment point represents 1-3measurements (mean ± SEM). Similar results were obtained in threeseparate liposome préparations.

thé pore appeared in thé présence of borate but not in itsabsence (Figure 2C), i.e. borate clearly enhanced thépalytoxin-induced pore formation. At 0-1-10 U.M palytoxin,pore formation could be seen without borate but was morepronounced in its présence (Figures 2D-F). Thus, as in cells,borate enhanced thé palytoxin effect in artificial membranes

Na,K-ATPase-mediated 86Rb-pumping and leak 249

24

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_ûcr.

16

12

8

A

0

20

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PYX1nM

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-B

PYX10jjM

min2 4 6

min

Figure 2. Borate enhances pore formation by palytoxin. The NKA-mediated uptake of 86Rb (O) was measured at 2, 5 and 7min;0 • 5 mM borate (B) was added from min 5 to 7 without effect on théion transport (A); 1 nM to 10 IIM palytoxin was then added at min 5with (•) and without (D) 0 • 5 mM borate and thé 86Rb-content of théliposomes measured at min 7 (B-F); 1-3 measurements perexpérimental point are shown together with SEM values. The NKA-liposomes were prepared and processed for 86Rb-uptake asdescribed in thé legend to Figure 1 and in Expérimental procédures.

containing isolated NKA molécules and was therefore présentin ail succeeding experiments.

Increasing palytoxin concentrations overcome ouabainantagonism

In thé left panel of Figure 3, thé increase in pore formationwith augmenting palytoxin concentrations is shown. In théabsence of palytoxin (A), 86Rb-uptake was measured at 2,5 and 8 min; thé 5-min value was set at 100%. When 100 nMpalytoxin was added from min 5 to min 7, -30% of théliposomes became leaky (B) as determined by thé decreasein liposomal 86Rb content occurring during removal ofexternal isotope by gel filtration at 0°C. The fraction did notincrease with additional incubation time (data not shown),indicating that it was limited by thé number of palytoxinmolécules available which is -3 toxin molécules per

en

mm mmFigure 3. Pore formation despite thé présence of ouabain byincreasing palytoxin concentrations. The uptake of 86Rb (O) wasmeasured at min 2, 5 and 7 (A); 100 nM (B), 1 HM (C) and 10 ^M (D)palytoxin (•) were added at min 5 and thé 86Rb leak determined atmin 7. Then, 100 JIM ouabain {•) was added at min 5 to inhibit théNKA and to block 86Rb-uptake; no further 86Rb uptake was observedin thé présence of ouabain (E); 100 nM (G) and 10 M.M (I) palytoxinwas then added at min 6 and thé 86Rb-leak measured at min 8;NKA-liposomes were prepared and processed for 86Rb-uptake asdescribed in thé legend to Figure 1. The results are averages(mean ±SEM) obtained in five separate liposome préparations; théisotope content measured at min 5 was normalized to 100% to permitcombination of thé results obtained in separate, and hence slightlydifférent préparations.

liposome or 1 -5 toxin molécules per right-side-out orientedNKA molécule. Over 90% of thé liposomes were leaky at1 U.M palytoxin (C) and 100% at 10 ^M palytoxin (D). In théright panel of Figure 3, thé total arrest of active 86Rb-uptakeby 100 HM ouabain and thé tightness of thé resultingouabain-NKA complex is documented: no leak occurs within3 min despite an inside-out 86Rb-gradient (E). In anotherexperiment, thé 86Rb-uptake was arresîed by ouabain for1 min and 100nM palytoxin was then added for 2minwhich did not modify thé tightness of thé liposomes, i.e.100 JIM ouabain totally prevented thé toxic effect of 100 nM

250 S. M. Anner and M. Moosmayer

palytoxin (F). Conversely, when 1 U.M palytoxin was added-1 Q% of thé liposomes became leaky within 2 min (G) and-40% at 10 U.M palytoxin (H). Thus, increasing palytoxinconcentrations are able to overcome thé antagonistic ouabaineffect.

Ouabain is unable to prevent pore formation inouabain-resistant purified NKA

Does ouabain antagonize thé palytoxin effect by interactionwith thé receptor for cardioactive steroids of thé NKAmolécule? To answer this question, thé 500-fold species-dependent variation of thé ouabain-sensitivity (Detweiler1967) was exploited. The NKA alpha-1 isoform isolated fromthé kidney reflects well thé species-dependent variation inouabain-sensitivity (Schônfeld étal. 1986). Thus, ouabaininhibits thé alpha-1 NKA from thé rabbit kidney with a half-maximal inhibitory constant (/C50)= 1 u-M as compared to thé100u.M of rat enzyme (Anner état. 1992). Thus, weinvestigated whether thé antagonistic effect of ouabain onthé dose-effect curves of palytoxin was lower in thé rat thanin thé rabbit enzyme. Figure 4A shows thé dose-effect curveof palytoxin on pore formation in thé présence and absenceof ouabain. In thé absence of ouabain, thé dose-effect curvefor thé palytoxin effect on thé rabbit enzyme shows an/C50 = 200nM. When ouabain had been added prier topalytoxin according to thé expérimental design shown inFigure 3, thé dose-effect curve was shifted to thé right andthé /C50 was increased -100-fold (Figure 4A). In apreliminary study using NKA-liposomes to test pore formationby palytoxin, 10 U.M palytoxin, i.e. a 50-fold higher concen-tration, was required due to thé higher Rb concentration{250 U.M) used (Rey et al. 1988); Rb or K ions are palytoxinantagonists (Habermann 1989).

Ouabain did not modify thé interaction of palytoxin with théouabain-resistant rat kidney enzyme (Figure 4B). Thus,sensitivity of NKA activity to ouabain is a prerequisite forouabain-inhibition of thé palytoxin-induced pore, i.e. botheffects must be due to spécifie interaction of ouabain withthé receptor for cardioactive steroids. In agreement with théresults obtained herein with purified enzymes, >1mMouabain was required to prevent palytoxin-induced K-releasefrom rat erythrocytes as compared to a /C50 = 2 - 1 JIM forrabbbit and 0 -3 JIM for human erythrocytes (Ozaki étal.1985).

Palytoxin inhibits thé enzyme activiîy of purified rénalNKA

Is thé palytoxin-induced pore associated with inhibition of théNKA activity? In fact, inhibition of ATPase activity by palytoxinhas been reported, although at higher concentrastions thanthose required for pore formation (Grellef a/. 1988, Yodaef al.1988, Habermann 1989). The démonstration of NKA inhibitionby palytoxin is much dépendent on thé expérimentalconditions. First, we had used thé linked-enzyme assay forcontinuous measurement of ATP hydrolysis: 1-2 |ig NKAprotein was preincubated in 10 ni with various concentrationsof palytoxin and 8 u,l was added to 1 ml palytoxin-free solutionin thé thermostatted cuvette (37°C) of a spectrophotometer.

140

120

10 9 8 7 6 5-Log [Palytoxin] , M

Figure 4. Ouabain sensitivity of NKA is required for thé antagonisticeffect of ouabain on pore formation. (A) To ATP-filled NKA-liposomeswhich had accumulated 86Rb for 5 min, increasing concentrationsof palytoxin (O) were added for 2 min and thé 86Rb-content of théliposomes was measured as illustrated in Figures 2 and 3. The poreformation occurred with a half-maximal activation constant(,4C50) = 200 nM. In another séries of expérimenta, 100 ^M ouabain(•) was added 1 min before thé palytoxin (O) as shown in Figure3; thé <4C50 increased 100-fold to 20 HM. The values are from fiveseparate liposome préparations and represent 2-5 measurementsper expérimental point with SEM values (B). The same experimentswere performed with liposomes reconstituted with ouabain-insensitiverat kidney NKA with (•) or without (O) ouabain added prior topalytoxin. The values represent one or thé average of twomeasurements obtained in two separate liposome préparations.

Negligible enzyme inhibition was apparent, indicating that thétoxin was immediately released from thé NKA receptor (datanot shown). By contrast, when thé ATPase activity was

Na.K-ATPase-mediated 86Rb-pumping and leak 251

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Figure 5. Inhibition of thé NKA activity by palytoxin. The ATPaseactîvity was determined by phosphate release for 30 min at 37°Cin a médium containing 1 ng protein/^l, 100 mM NaCI, 5 mM MgCI2,1 mM EDTA and 25 mM imidazole, pH 7-4. Each expérimental pointrepresents thé mean of 3-6 measurements (mean ± SEM). The IC50

for enzyme inhibition -4 M.M.

measured by colorimetric phosphate détermination in théprésence of palytoxin, ATPase inhibition was apparent withan /C50-4 U,M (Figure 5); at 10 U.M palytoxin, thé enzymewas not yet fully blocked but higher palytoxin concentrationswere not used for technical reasons. The /C50 for enzymeinhibition is 20 times higher than thé IC50 of 100 nM foundfor pore formation (Figure 4). This différence could mean thatpore formation précèdes enzyme inhibition. Conversely, théprocess of pore formation in liposomes is différent fromenzyme inhibition by palytoxin since one pore per liposomeis sufficient for release of thé entrapped Rb ions. Thus, if aliposome contains several NKA molécules on average, onlyone of them needs to be opened by palytoxin for total 86Rbleakage to occur. By contrast, ail NKA molécules must beinactivated for full ATPase inhibition.

Discussion

Palytoxin, isolated form thé coral family Palythoa, is thé mostpotential animal toxin isolated to date; its high toxicityoriginates in its ability to create leaks in thé cell membrane.NKA has been proposed as a target System since ouabain,a spécifie NKA inhibitor, antagonizes thé palytoxin-inducedpore formation in cells (Habermann et al. 1 981 , Ishida et at.1981). However, thé cell membrane contains numeroustransmembrane proteins and receptors, hence palytoxincould bind to a spécifie receptor which, in turn, may interactwith thé NKA System. Therefore, thé purified transport Systemwas inserted in a functional state into artificial membranes.When NKA was contained in thé membrane, a palytoxin-induced pore appeared as indicated by leakage of 86Rb fromATP-filled liposomes which had previously accumulatedexternal 86Rb ions by thé ouabain-sensitive transport activityof thé pump.

It is very likely that palytoxin unmasks a naturalconductance pathway normally contained in thé NKA System.In fact, when in previous experiments NKA-liposomescontaining one pump molécule were fused with a planarbilayer, single channel measurements revealed a 40-50 pSconductance channel at a +20 mV cisltrans voltage;subevents of about 10 pS appeared regularly (Reinhardt et al.1984). Interestingly, this value is close to thé conductanceof thé channel induced by palytoxin in cell membranes whichranges from 8 • 9 to 9 • 6 pS as measured by patch clamping(Muramatsu et al. 1988, Kim et al. 1991, Kinoshita et al. 1991,Sauviat 1992). The channel is perméable to K, Na, Li, cholineand guanidine (Castle and Strichartz 1988, Sauviat 1992).

How does palytoxin modify thé sodium pump cycle? Thevectorial translocation of Na and K by thé NKA Systemcomprises a fundamental E1-E2 change as depicted in thésimplified scheme of thé pump cycle (Scheme 1). Uponinteraction with Na and ATP, a phosphorylated E1-conformation is formed. In thé présence of K ions, an E1-Pto E2-P transition occurs, followed by dephosphorylation andreturn tothe E1-conformation. Palytoxin is most potent in théprésence of ATP and in thé absence of K ions (Habermann1989), i.e. in a condition favouring thé E1-P conformation(Scheme 1). Palytoxin could inactivate a permeability barrierwhich normally protects thé ion transport pathway containedin thé pump System; such a barrier must prevent backleakor slipping of ions during thé ATP-driven Na,K-antiportprocess.

Conversely, ouabain can interact with thé E2- (Scheme 1)as well as with thé E^form (Wallick étal. 1978). Whenouabain is added to thé ATPase in turnover conditions, i.e.in thé présence of ATP, Na and K ions, it stabilizes anE2-conformation as visualized by differential trypsinolysis(Anner et al. 1994); this E2-ouabain conformation,reconstituted into liposomes, was found to be imperméable

E -palytoxin.••'•; (channel)

palytoxin1 ^"CV

^ATP J

\^^s F

1 r

rP ,:'..... ouabain

E 2 -ouabain

Scheme 1. Localization of palytoxin and ouabain action in thé pumpcycle. In thé présence of N and ATP thé E1-conformation isphosphorylated to E1P. This form has high affinity to palytoxin.Ouabain may interact with thé E1- and E2-forms and stabilize théenzyme in a dead-end E2-ouabain conformation which isimperméable to K ions, in contrast to a ouabain-free E2-conformationformed in thé absence of ATP.

252 S. M. Anner and M. Moosmayer

to K ions, in contrast to thé ouabain-free E2 conformation.Thus, palytoxin stabilizes a leaky E1-conformation andouabain a tight E2-conformation. The palytoxin-ouabainantagonism would then be due to allosteric interaction of thétwo toxins via their respective receptors, thé potent animaltoxin pulling thé NKA towards a leaky state and thé weakerplant toxin ouabain pushing it to a tight state. Thus, a planttoxin might protect thé System from thé distinct action ofanother, even more potent, animal toxin by conformationalallosteric compétition.

Ouabain antagonizes thé palytoxin-induced pore formationin artificial membranes reconstituted with ouabain-sensitiveNKA but not in présence of ouabain-resistant NKA. Theconformational model proposed in thé présent studyprésumes that thé antagonism between thé two toxins doesnot necessarily require interaction of both compounds withone and thé same receptor site. Palytoxin has been proposedto share thé ouabain receptor at least partially since onlyglycosylated cardioactive steroids are antagonistic whereasthé aglycones are inefficient; glycosides containing threesugars such as digoxin and digitoxin are also less efficientalthough they inhibit thé ATPase activity as potently as doesouabain (Ozaki et al. 1984, 1985). Perhaps thé dissociationrate of thé compounds plays a rôle: thé faster thé dissociationrate, thé lower their ability to antagonize palytoxin action. Toexplain thèse observations by thé conformational allostericmodel, one must consider thé fact that thé less tightly boundcompounds are unable to induce a stable inhibitor-NKAcomplex. Thus, in terms of thé allosteric interaction model,either thé aglycone-induced E2 conformation or thé ouabain-induced conformation in ouabain-resistant enzymes wouldbe unstable ligand-receptor complexes and hence unable toprevent thé palytoxin-induced shift to thé leaky E1conformation.

In cells, palytoxin produces a rapid loss of thé intracellularK followed by Ca-influx leading to death by variouscardiovascular and neurological symptoms resulting from théionic disbalance (Habermann 1989). Palytoxin acts also asa non-phorbol tumour-promoting agent by its ability toenhance Na-influx (Wattenberg étal. 1989) which couldactivate growth-promoting factors (Repke 1988) and modulategène expression (Nakagawa étal. 1992). By contrast, thécardioactive steroids are able to induce differentiation oftumour cells (Zimmermann and Speers 1987, Zhang et al.1991). An increase in thé intracellular Na/K ratio is a primarysymptom of many viral infections, be it by viral NKA inhibitionor by a virus-induced Na/K-leak (Del Castillo étal. 1991).Palytoxin-derivatives with lowered toxicity (Tosteson et al.1991) could be used to cover and protect thé extracellularpart of thé NKA from attack by chemicals, enzymes andviruses.

Expérimental procédures

Materials

Palytoxin from Palythoa caribaerum was kindly provided by Prof. E.Habermann, Justus-Liebig University, Giessen, Germany. Cholic acidand EDTA (Tilriplex II) were purchased from MerckABS (Zurich, CH);phosphatidylcholine grade Ha and phosphatidylsehne were from LipidProducts (Nutfield, UK); cholestyramine-resin was from Serva (Basle,

CH). 86RbCI was obtained from Amersham International(Amersham, UK); Salts and buffers were of analyticat grade; onlybi-distilled water was used.

NKA purification and acîiviîy measurements

NKA was purified from thé outer medulla of rabbit or rat kidneys,thé protein determined and thé activity measured by thé linked-enzyme assay as described in Anner et al. (1994). For measurementof palytoxin inhibition of NKA activity, thé enzyme activity wasdetermined by phosphate release by thé highly sensitive colourimetricmethods of Chen et al. (1956) as modified by Âmes and Dubin (1960).

Concept and préparation of two-sîded bi-dîrectionalNKA-liposomes

A new System for analysing thé extracellular receptor component(s)of purified actively transporting NKA molécules has been developed:two-sided ATP-filled NKA-liposomes. The motivation for thisdevelopment was thé need for a model containing purified, transport-active NKA molécules which expose their receptor for cardioactivesteroids at thé liposome surface in order to be able to test thé isolatedendogenous NKAinhibitorsof unknown structure (Anner et al. 1990,Anner and Haupert 1993). The model was conceived on thé basisof extensive functional, statistical and mathematical analysis ofmorphologically characterized NKA-liposomes (Anner 1985). TheNKA-liposomes hâve been desribed structurally and functionally byextensive studies which led to thé characterization of (i) thé averagesize(100 nmjand size distribution of thé liposomes, (ii) thé numberof liposomes per millilitre (2x1013) corresponding to a concentrationof -3x10~8 M, and (iii) thé liposome-to-liposome distance of about240 nm, (iv) thé average number of four reconstituted NKA moléculesper liposome at thé lipid/protein ratio selected for thé experiments,corresponding to a concentration of about 1 -2x10~ 7 M and (v) théknowledge of thé random orientation of thé reconstituted molécules(Anner et al. 1984ab). The fact that 50% of thé NKA molécules werefound in right-side-out cellular orientation (Anner et al. 1984ab) wasexploited to develop a System in which NKA functions as in thé cell,with ATP and Na inside thé liposomes.

Functional NKA was reconsitituted randomly into ATP-filledliposomes as previously described (Anner and Mcosmayer 1985).Briefly, 180|ig purified NKA was suspended in 60 (il 50 mMNa2ATP, 30 mM histidine, 1 ITIM Tris-EDTA, 5 mM MgCI2 and 23 HIMNa cholate; thé pH was adjusted to 7-10 by thé addition of-120 mu NaOH, 23 mM cholic acid, pH 7-2, 0°C. The supernatantresulting from a 10-min centrifugation at 100000g in a BeckmanAirfuge was added to 50 ni 5 mM MgCI2, 30 mM histidine, 1 mM Tris-EDTA, 23 mM cholic acid, 0-8 mg phosphatidylcholine and 0-2 mgphosphatidylserine, pH 7-2, 0°C. The lipid solution was preparedaccording to previously published procédures (Anner and Moosmayer1981). ATP-filled liposomes containing on average -10000molécules per liposome were formed by removing thé détergent for15 h at 0°C in 5 ml 50 mM Na2ATP, 30 mM histidine, 5 mM MgCI2,1 mM Tris-EDTA and 200 mg washed cholestyramine resin (pH 7-2),0°C. The pH was adjusted by adding 120 mM NaOH ; together withthé 100 mM Na added with 50 mM Na2ATP, thé total Naconcentration was 220 mM. The external ATP was removed by twocentrifugations in thé Beckman Airfuge for 70 min at 100 000 g at0°C in a solution containing 100 mM NaCI, 5 mM MgCI2, 30 mMhistidine and 1 mM Tris-EDTA, pH 7-2, 0°C. The osmotic differrencedue to thé 220 mM internai Na versus 100 mM external NaCI did notinfluence thé transport results due to thé ion-imperméable bilayerand thé high osmotic stability of thé liposomes. The préparationswere stable at 0°C for 48 h as tested by active 86Rb uptake (Reyet al. 1987). The protein content was -0-5 mg protein/ml.

Successive measurement of 86Rb-accumulation,inhibition and leak by gel filtrationAddition of micromolar concentrations of external 86Rb induces slow86Rb-uptake which proceeds linearly for minutes and establishes upto 30-fold transmembrane inside-out Rb-gradients (Rey et al. 1987).

Na,K-ATPase-medialed 86Rb-pumping and leak 253

Scheme 2. (A) 86Rb-accumulalion by NKA in right-side-out(cellular) orientation in ATP-filled liposomes. (B) Palytoxin forms aleak through thé NKA molécule and thé entrapped 86Rb ions leakout during thé gel filtration step used for thé removal of external86Rb (C). When ouabain is added, 86Rb uptake is blocked (D).Palytoxin is not able to interact with thé ouabain-blocked pump (E)and no leak is formed; thus ail thé 86Rb remains entrapped (F).

A radioactive solution (3 ni) containing 20 JIM carrier-free 86RbCI,100 rriM NaCI, 5 mM MgCI2, 1 ITIM Tris-EDTA and 30 mM histidine,(pH 7-2) was added to 3 ni ATP-filled liposomes and incubated for2-10 min at 25°C {cell-like 86Rb-uptake by right-side-out orientedpumps). Up to 30% of thé total 86Rb werre pumped into liposomesby thé right-side placed pumps within 8min (Rey étal. 1987).Ouabain and palytoxin were added as indicated in thé figure legends.Transport was stopped by adding to thé sample 120 ni ice-cold stopsolution (100 mM NaCI, 5 mM KCI, 30 mM histidine, 1 mM Tris-EDTA,pH 7-2); 10 ni aliquots were counted in duplicatesto détermine thétotal amount of 86Rb added and 100 jil aliquots were put on top ofa 1x20 cm Sephadex G-50 médium column. The column was elutedwith stop solution at 0°C and thé washed liposomes were collectedwithin thefirst 10 min at a flow rate of 0 -8 ml/min. The radioactivityassociated with thé washed liposomes was measured by scintillationcounting and expressed as a percentage of thé total radioactivityadded to thé liposomes.

The séquence of events is îllustated in Scheme 2. First, additionof external ̂ Rb to thé liposomes containing randomly reconstitutedNa,K-ATPase molécules induces active 86Rb accumulation (A).Externally added palytoxin interacts with thé extracellular receptorof thé right-side-out oriented Na,K-ATPase molécules (B) leading topore formation and leakage of thé internai 86Rb during liposomewash by gel filtration (C). By contrast, externally added ouabainstabilizes thé enzyme in a tight form (D). Ouabain antagonizes thépalytoxin interaction with ils receptor (E) and thé liposomes retaintheir internai 86Rb during thé washing procédure (F). A relativelyhigh ouabain concentration of 100 ^M was used in ail experimentsto obtain rapid and complète inhibition of 86Rb-uptake in additionto efficient inhibition of palytoxin action.

AcknowledgementsWe are grateful to Prof. Ernst Habermann for thé gift of palytoxin,and for many helpful discussions and advice concerning théexperiments and thé manuscript. We thank Drs A. De Pover andR. M. Anner for reading thé text and Mr F Pillonel for skilful artwork.Supported by thé Swiss National Science Foundation Grant Nos3.535-0.83, 3.502-0.86 and 31-25666.88 (B.M.A.).

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Received 2 February 1994, and in revised form 25 July 1994,