The EMBO Journal vol.7 no.1 1 pp.3353-3359, 1988

On the membrane translocation of diphtheria toxin: atlow pH the toxin induces ion channels on cells

Emanuele Papini, Dorianna Sandona,Rino Rappuoli' and Cesare Montecucco

Centro CNR Biomembrane e Dipartimento di Scienze Biomediche,Universita di Padova, Via Loredan 16, 35131 Padova and 'CentroRicerche SCLAVO S.p.A., Via Fiorentina 1, 53100 Siena, Italy

Communicated by G.Cesareni

Diphtheria toxin (DT) in acidic media forms ion-conducting channels across the plasma membrane andinhibits protein synthesis of both highly and poorly DT-sensitive cell lines. This results in loss of cell potassiumand in entry of both sodium and protons with aconcomitant rapid lowering of membrane potential. ThepH dependency of the permeability changes is similar tothat of the inhibition of cell protein synthesis. DT-inducedion channels close when the pH of the external mediumis returned to neutrality and cells recover their normalmonovalent cation content. Similar permeability changeswere induced by two DT mutants defective either inenzymatic activity or in cell binding, but not with amutant defective in membrane translocation. Theimplication of these findings for the mechanism of DTmembrane translocation is discussed.Key words: diphtheria toxin/ion channels/monovalentcations/membrane potential

Introduction

Diphtheria toxin (DT) is a very powerful protein toxinresponsible for clinical diphtheria (Pappenheimer, 1981). Itis produced by Corynephago (tox+) infected Coryne-bacterium diphtheriae as a single chain (mol. wt 58 342 kd)whose sequence has been determined (Greenfield et al.,1983; Ratti et al., 1983). The protein is cleaved by proteasesinto two fragments connected via a disulphide bridge: theA chain (21.164 kd) is an ADP-ribosylase, while chain B(37.194 kd) is involved in cell binding (for reviews see:

Olsnes and Sandvig, 1985; Ward, 1987).DT belongs to the group of bacterial protein toxins with

intracellular targets (Middlebrook and Dorland, 1984). Theirmechanism of cell intoxication can be conveniently dividedinto three main steps: (i) cell binding, (ii) membranetranslocation and (iii) cytoplasmic target modification. Thereis evidence that both a plasma membrane protein(s) andphospholipids are involved in the binding of DT to cells(Moehring and Crispell, 1974; Alving et al., 1980; Olsneset al., 1985; Cieplak et al., 1987; Papini et al., 1987a). Thethird step of DT action involves the ADP-ribosylation,catalysed by fragment A, of elongation factor 2 withconsequent block of protein synthesis (Collier, 1982; Ward,1987).The second step is the least understood. Since toxin binds

to the cell surface and intoxication occurs in the cytoplasm,

©IRL Press Limited, Oxford, England

the water-soluble DT molecule must somehow cross thehydrophobic membrane barrier. Available evidence indicatethat DT enters the cytoplasm from a low pH compartment(Olsnes and Sandvig, 1985). Experiments performed witha variety of techniques have shown that DT undergoes a low-pH-driven conformational change that occurs in a range ofacidic pHs overlapping that found in endosomes (Sandvigand Olsnes, 1981; Blewitt et al., 1985; Montecucco et al.,1985; Papini et al., 1987b,c). This structural change resultsin the exposure of hydrophobic surfaces that enable the toxinto enter in contact with the hydrocarbon chains ofphospholipids and detergents (Sandvig and Olsnes, 1981;Hu and Holmes, 1984; Zalman and Wisnieski, 1984;Montecucco et al., 1985; Papini et al., 1987a,c).At low pH, DT forms ion-conducting channels across

planar lipid bilayers (Donovan et al., 1981; Kagan et al.,1981; Hoch et al., 1985). Based on the results obtained withthis model system, a mechanism has been suggested for theentry of DT into cells. It was proposed that at acidic pHsfragment B forms a transmembrane tunnel large enough toaccommodate the A chain in an extended form. Thehydrophilic A chain unfolds at low pH and transverses themembrane inside the tunnel, shielded from the contact withthe hydrocarbon tails of lipids (Kagan et al., 1981; Hochet al., 1985). Even though this tunnel model does notaccommodate some recent observations, it offers aninteresting hypothesis for the little-understood process ofprotein membrane translocation: that it is not limited totoxins, but occurs in cells for all those proteins importedfrom the cytoplasm into organelles (Zimmerman and Meyer,1986; Eilers and Schatz, 1988).In the present work we have obtained evidence for the

formation of DT ion channels also in living cells during theprocess of cell intoxication. Cells were treated with DT atacidic pHs in order to introduce the toxin from the plasmamembrane (Sandvig and Olsnes, 1980, 1981; Draper andSimon, 1980). The effects of DT on plasma membranepermeability and on protein synthesis of both highly andpoorly DT-sensitive cells were investigated and comparedwith those caused by some DT mutants that cross-reactimmunologically with DT but are defective either in cellbinding (crm 45) or in membrane translocation (crm 1001)or in enzymic activity (crm 197).

ResultsModifications of K+ and Na+ cellular contentsinduced by DTStudies with model systems have shown that at acidic pHsDT forms ion channels across lipid bilayers (Donovan et al.,1981; Kagan et al., 1981; Miesler, 1983; Zalman andWisnieski, 1984; Hoch et al., 1985). Here we have testedthe possibility that DT ion channels also assemble on theplasma membrane of living cells during cell intoxication by

3353

E.Papini et al.

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Fig. 1. Effect of DT on cation permeability and protein synthesis of cells. Vero (A) and CHO (B) cells were incubated for 1 h at 4°C as describedin Materials and methods. Na+ (E-El) and K+ (0-0) cellular contents were determined by flame photometry after a 15-min incubation with DTat 37°C, pH 4.9. Protein synthesis (A-A) was estimated as [14C]leucine incorporation after 5 min at pH 4.9, because at this time intoxication isnearly maximal and prolonged exposure to low pH leads to a decrease in the amount of leucine incorporated by controls. On the other hand, K+ andNa+ were measured after 15 min to have a better chance to trace their fluxes. As the control value of intracellular Na+ concentration is difficult toestimate because of residual extracellular contamination, the content of this cation was expressed as an increment with respect to controls.K+ contents and Na+ increments were referred to a control cell K+ concentration of 140. Points are averages of at least four different experimentsrun in triplicate and bars represent standard deviations.

measuring the K+ and Na+ content of cells after incubationwith the toxin at low pH. If pores are assembled on theplasma membrane, potassium ions should leak out whilesodium ions should enter into the cell.

Figure IA shows that the K+ and Na+ content of Verocells, incubated for 1 h at 4°C with DT, washed and thenexposed to pH 4.9 for 15 min at 37°C, changes as a functionof DT concentration. In the presence of 10-7 M DT, theintracellular concentration of potassium is reduced from

- 140 mM (Arosen, 1985) to -40 mM while that ofsodium increases from the basal value of - 9 mM (Arosen,1985) to - 125 mM; low pH alone has no effect (notshown). Na+ influx and K+ efflux are already detectableat toxin concentrations as low as 6 x 10-10 M and theratio between the two fluxes is always > 1, indicating thatsodium uptake is more pronounced than potassium efflux.

Figure lA also shows that the dose-response curve ofthe inhibition of protein synthesis, measured under the sameconditions, is shifted by at least one log unit toward lowertoxin concentrations as compared to those relating to ionfluxes; with 10-10 M DT, protein synthesis is 90%inhibited while the effect on K+ and Na+ levels begins tobe detectable. Ouabain does not modify these patterns (notshown) indicating that the shift between the cytotoxic andpermeability effects is not due to a compensatory activationof the Na+/K+-ATPase.The above-described results were obtained with Vero cells,

a line highly sensitive to DT with a number of high-affinityDT receptors estimated to be - 105/cell (Middlebrooket al., 1978; Mekada et al., 1982). However DT ionchannels were observed in lipid model systems lacking suchreceptors. Hence investigations were extended to CHO cellsthat are poorly sensitive to DT because they possess

- 100-fold less high-affinity receptors per cell (Mekada

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Fig. 2. Effects of DT on non-washed CHO cells. Cells were incubatedwith DT at 37°C and immediately afterwards pH was dropped to 4.9with (closed symbols) or without (open symbols) 0.75 mM amiloride.Na+ (Eli-K) and K+ (0-0) cellular content and protein synthesisinhibition (A-A) were determined as described in the legend toFigure 1. Values reported are the average of four separate experimentsrun in triplicate and bars represent standard deviations.

et al., 1982). Figure lB shows that also under the presentconditions CHO cells are less sensitive than Vero cells witha protein synthesis inhibition curve shifted toward muchhigher DT concentrations. The shift is closely related to thedifference in the number of DT receptors between the twocells lines. None the less, even in this case, DT-induced ion

3354

Membrane permeability changes induced by diphtheria toxin at acidic pH

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Fig. 3. Permeability and cytotoxic effects induced in CHO cells by crm 45 and crm 1001. Protein synthesis inhibition (A) and monovalent cations

fluxes (B) induced by DT ( ), crm 45 (------) and crm 1001 (. ) measured as described in the legend to Figure 2. Points are averages of

two experiments run in triplicate; standard deviations are shown as bars.

channels are present and again there is a shift between thedose dependency curves of protein synthesis inhibition andthe permeability changes to monovalent cations.The effects of DT are to be ascribed to toxin molecules

bound to the cell surface since unbound toxin is washed away

before the addition of the acidic medium. On the other hand,in most studies performed with model membranes, unboundtoxin is present in the medium at the time of acidification.The two situations may be different since it is known thatDT aggregates in acidic solutions and such DT aggregatescould be responsible, upon partition into the lipid bilayer,for the formation of ion-conducting channels. Hence we havetested the effects of DT on cells under conditions similarto those used with artificial membranes. DT was added ina physiological buffer at pH 7.4 to CHO cells at 37°C inthe presence of 10 mM NH4Cl, to inhibit DT entrance inthe cytosol from intracellular acidic compartments, and thepH of the medium was lowered by adding an aliquot of acidicbuffer. Figure 2 shows that under these conditions there isan increase in protein synthesis inhibition (- 1 log unit)accompanied by a much larger effect on the permeabilityto monovalent cations ( 2 log units). A similar finding wasobtained with Vero cells (not shown).An increased ability of DT to intoxicate unwashed cells

is expected on the basis of the higher amount of toxin present;however, the higher effect on permeability than on proteinsynthesis indicates that, under such conditions, DT can beassembled into forms that are more efficient in increasingmembrane permeability than in translocating fragment A.

Figure 2 also shows that the presence of amiloride, an

inhibitor of Na+/H+ and Na+/Ca2+ antiporters (Smithet al., 1982; Aronsen, 1985), does not affect K+ and Na+fluxes, thus suggesting that Na+ influx is not due toactivation of such sodium antiporters.

Effect of DT mutants on the cellular permeability tomonovalent cationsCrm 45 lacks a 12-kd C-terminal segment of the B fragment(Giannini et al., 1984), binds to cells with low affinity

(Boquet and Pappenheimer, 1976), forms channels across

planar lipid bilayers (Kagan et al., 1981) and inserts intolipid bilayers as well as DT does (Papini et al., 1987b,c).Figure 3 shows that when the medium of CHO cells was

acidified in the presence of crm 45, cellular protein synthesiswas inhibited in a range of toxin concentrations - 100-foldhigher than with DT. As with DT, changes in the cellcontents of K+ and Na+ are detectable only at toxinconcentrations where protein synthesis is nearly completelyinhibited.Crm 1001 has a single amino acid substitution (Cys-471

replaced by Tyr) (G.Ratti et al., unpublished results), bindsto cells as well as DT does and has a similar enzymaticactivity (Zucker, 1983). However, it has a very low toxicitiybecause it is defective in membrane translocation (Papiniet al., 1987c). Also, under the conditions used here crm

1001 is poorly toxic (Figure 3). Moreover, at concentrationsas high as 10-7 M it does not induce any change in thepermeability of CHO cells to monovalent cations. Hencethere appears to be a correlation between the ability of DTto translocate its A fragment in the cytoplasm and that ofinducing the formation of ion channels. Moreover, this resultis consistent with the idea that the DT-induced changes ofpermeability are due to channels formed by the toxin andnot by another cell protein activated by the toxin.Crm 197 is non-toxic because of a single mutation in the

A fragment (Gly-52 replaced by Glu) (Giannini et al., 1984)that abolishes the NAD-glycohydrolase activity of the toxin;on the other hand it inserts into membranes at acidic pHsas well as DT does (Papini et al., 1987a). Crm 197 altersthe permeability of Vero cells to monovalent cations in a

manner comparable to that of DT but without any effect onprotein synthesis (not shown). This experiment shows thatthe inhibition of protein synthesis found above is not to beattributed to the alteration of permeability brought about byDT and that this latter effect is reversible (see below).Moreover, it offers the possibility of using crm 197 as a

reagent to quench the transmembrane pH gradient ofendosomes.

3355

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Fig. 4. pH dependence and reversibility of DT-induced changes in K+ and Na+ cellular contents of CHO cells. Panel A shows the pH dependenceof the inhibition of protein synthesis of CHO cells incubated as described in the legend to Figure 2 with 5 x 10-8 M DT for 5 min (A-A). PanelB reports the pH dependence at 0, 5 and 15 min of K+ (0-0) and Na+ (0-0) cellular contents of CHO cells incubated with the same amountof DT at the indicated pHs. Panel C: K+ and Na+ cellular content of cells incubated with DT for 15 min at the indicated pHs, washed and thenfurther incubated for the periods indicated at 37°C in MEM medium, pH 7.4. Symbols as in Figure 1; standard deviation bars are omitted forclarity.

Taken together, the results obtained with DT mutantsindicate that the change in permeability is caused by theN-terminal 25-kd part of fragment B in agreement with theresults obtained with planar lipid bilayers (Kagan et al.,1981; Miesler, 1983).

pH and time dependence and reversibility of DT-induced cellular modificationsTo be relevant to the process ofDT membrane translocation,the pH dependence of the DT-induced change of permeabilityshould fall within the range of pHs found in endosomes(Mellman et al., 1986). Figure 4 A and B shows that thecellular effects of DT are maximal already at pH 5 and thatabove pH 5 the effects of DT on protein synthesis and onthe cellular content of sodium and potassium have a similarpH dependence. Figure 4C also shows that the inhibitionof protein synthesis is not due to a membrane lytic effectof DT because the normal cellular content of sodium andpotassium can be regained by cells after a short recoveryperiod.As found above, the two phenomena induced by DT are

not comparable because the permeability to both K+ and toNa+ rapidly decreases below pH 5, while the inhibition ofprotein synthesis remains at the same level. Such a bell-shaped pH dependence of permeability could reflect theexistence, at pHs lower than 5.0, of a toxin state less effectivein inducing cation transmembrane fluxes although still ableto transfer the A chain into the cytosol; or could result froma conversion of DT channels from a cation to an anionselectivity. Such conversion has been shown to occur onplanar lipid bilayers, though at more acidic pHs (Hoch et al.,1985).The uptake of sodium and release of potassium are both

rather slow. The change in cellular monovalent cation contentstarts seconds after acidification and continues for at least

Ap moles

ofB

pH4.9

l/GRAM

- 1.0

- 0.5

- 0.2

- 0.0GRAM

10min

Fig. 5. DT-induced K+ efflux from Vero cells followed with aK+-selective electrode. (A) Trace recorded with a potassium-selectiveelectrode immersed in a flask of Vero cells incubated for 15 min with5 x 10-8 M DT, washed and the flask, filled with an isotonic cholinemedium pH 4.9, mounted vertically on a magnetic stirrer. The dottedtrace corresponds to cells treated in the same way without the toxin.4 ttg/mn of gramicidin D was added where indicated. (B) Trace from asimilar experiment performed in a calcium-free medium. The signalproduced by known K+ amounts is shown on the right side.

15 min (Figure 4A); more prolonged exposures to low pHcould not be tested because these start to cause irreversiblecell damage. Taken together, the present findings are inagreement with the idea that at low pH DT forms acrossthe plasma membrane ion-conducting channels that remainopen as long as a transmembrane pH gradient is present;when external pH is returned to neutrality, channels closeand the cell rapidly re-establishes a normal ion content beforeany effect consequent to inhibition of its protein synthesiscan be noticed.

3356

Membrane permeability changes induced by diphtheria toxin at acidic pH

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Fig. 6. Low pH DT-induced depolarization of cells detected with afluorescent probe. Traces show the change of fluorescence of themembrane potential probe diS-C3(5) after uptake by Vero cells andmouse spleen lymphocytes following acidification of the medium to pH4.9 in the presence of different concentrations of DT (reported beloweach curve with 8% phosphoric acid. Gram indicates the addition ofgramicidin D (4 jig/ml) and a-toxin the addition of S.aureus (x-toxin(0.5 ztM final concentration). Fluorescence is reported in arbitraryunits.

DT-induced K+ release measured with a potassium-selective electrodeThe effiux of intracellular potassium caused by DT at acidicpH was confirmed by following its appearance in theextracellular medium. We have tested both spleen lympho-cytes resuspended in choline medium and CHO or Vero cellsadherent to the wall of a plastic bottle. Figure 5 shows therecord from a K+-sensitive electrode immersed in themedium bathing a monolayer of Vero cells covering one wallof the flask. The kinetics and pH dependence (not shown)of the K+ release induced by DT are very similar to thoseobtained by measuring cellular ion contents by flamephotometry. The absence of calcium (panel B) does notmodify the K+ efflux and, since no sodium ions were pre-sent in the medium, it is very unlikely that potassium isreleased from cells in conjunction with sodium or calciuminfluxes. On the other hand, the possibility that K+ effluxis due, at least in part, to a proton influx that activates aDT-independent K+ efflux pathway cannot be excluded.Similar results were obtained with CHO cells and with mousespleen lymphocytes (not shown).

DT at acidic pHs lowers membrane potentialThe fluorescent probe diS-C3(5) has been extensively usedto follow changes in membrane potential (Waggoner, 1976;Tsien and Hladky, 1978; Rink et al., 1980). This mero-cyanine dye is taken up by cells in a membrane-potential-dependent process and the binding to cell structures leadsto a quenching of its fluorescence. Depolarization causes dyerelease and a consequent recovery of fluorescence. Figure6 shows the decrease in dye fluorescence accompanying its

cell uptake. Acidification causes a fluorescence increase dueto a direct effect on dye fluorescence intensity and to adepolarizing effect on protons (Sandvig et al., 1986). Despitethese effects, that lower the sensitivity of this assay, it clearlyappears that DT at low pH is able to depolarize fibroblastsand lymphocytes further. Again its effect appears to be ratherslow since gramicidin added after DT caused a furtherincrease in fluorescence. For a comparison, Figure 6 alsoreports the effect on membrane potential of the a-toxin ofStaphylococcus aureus, which causes haemolysis and formslarge pores across the plasma membrane (Bhakdi et al.,1981; Menestrina, 1986).

Discussion

Membrane translocation is the least understood step in theprocess of cell intoxication by toxins with intracellulartargets; somehow a water-soluble molecule such as DTbecomes able to cross the hydrophobic membrane barrier.This passage is common to a variety of proteins that aresynthesized in the cytoplasm and are to be localized in innercompartments of cell organelles such as endoplasmicreticulum, mitochondria and chloroplasts (Zimmermann andMeyer, 1986; Eilers and Schatz, 1988). The first reportsbearing on the membrane translocation of DT showed thatDT causes a pH-dependent increase in the conductance ofplanar lipid bilayers (Donovan et al., 1981; Kagan et al.,1981).In the present paper we show that DT at acidic pHs alters

the plasma membrane permeability to monovalent cationsof cells. The simplest interpretation of these results is thatDT at low pH forms ion-conducting channels in living cells,resulting in potassium efflux and sodium and proton influxwith a consequent decrease of membrane potential. This issupported by the finding that crm 1001 binds to cells butdoes not cause any change in permeability. However, wecannot exclude the possibility that at least part of the fluxesoccur via a DT-independent mechanism activated byacidification.

It has been suggested that at low pH fragment B formsa transmembrane tunnel that allows the passage of theenzymatic fragment A in an extended form (Kagan et al.,1981; Hoch et al., 1985). In this model the formation ofa large ion-conducting pore is inherent to the process offragment A entry into the cell and hence the two phenomenashould be related.Here we present evidence that there is a range of DT

concentrations that cause a large inhibition of proteinsynthesis without a measurable effect on the cellular contentof monovalent cations; when 90% or more of Vero and CHOcells protein synthesis was inhibited, - 20 A fragments hadpenetrated into the cytoplasm during the 5 min of low pHincubation with a very small, or non-measurable, effect oncell sodium even after 15 min. Moreover, the DT-inducedchange in cell potassium and sodium is rather slow, and thisis unexpected on the basis of the conductance measured inplanar lipid bilayers. Assuming that DT forms pores ofsimilar conductance in model and cellular systems, one canestimate that sodium ions should have equilibrated acrossthe plasma membrane in - 15 min. This discrepancybetween artificial and cellular systems cannot be ascribedto DT receptors because similar results were obtained with

3357

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cells differing in their number of toxin receptors and alsowith crm 45 which is unable to bind to cells with highaffinity. Indeed recent evidence indicates that DT receptorsare needed for driving the toxin into endosomal compart-ments, but not for its translocation into the cytoplasm(Colombatti et al., 1986; Greenfield et al., 1987; Murphyet al., 1986; Bacha et al., 1988).The simplest explanation for the difference between

cellular and model systems is that DT ion channels assembledat low pH on cells have either a lower mean conductanceor are closed for most of their life-time.Some results have been recently reported that cannot be

easily accommodated in a protein tunnel model. Sandviget al. (1986) have shown that the reduction of cell membranepotential does not lower the ability of DT to translocate itsenzymic A part in the cytosol, while DT ion channels acrossplanar lipid bilayers are voltage dependent (Donovan et al.,1981; Kagan et al., 1981). Moreover, hydrophobicphotolabelling experiments have shown that at low pH bothfragment B and fragment A interact with the fatty acid chainsof phospholipids (Hu and Holmes, 1984; Zalman andWisnieski, 1984; Montecucco et al., 1985; Papini et al.,1987c) and that the pH dependence of this phenomenon isclosely related to that of DT intoxication of cells subjectedto a low pH pulse (Sandvig et al., 1986; Papini et al.,1987c).The presently available data can be better fitted to a cleft

model for the membrane translocation of fragment A (Bissonand Montecucco, 1987; Papini et al., 1987c; Singer et al.,1987). In this model it is envisaged that, at low pH, a Bfragment(s) inserts in the lipid bilayer, with its hydrophobicsurfaces exposed to lipids, and forms a hydrophilic cleft thatfaces the hydrophilic residues of an A fragment. At variancewith the tunnel model, the A fragment remains in contactwith lipids via its hydrophobic residues during its membranetranslocation. The matching of hydrophilic and hydrophobictypes of interactions between lipids, A chain and B chainwould reduce the energetic cost of the process as comparedto the tunnel model where the hydrophilic B channel has toaccommodate not only the hydrophilic residues of the Achain but also the 68 hydrophobic residues present in its193-residue-long sequence. After refolding and release ofthe A chain in the cytoplasm by disulphide reduction, thehydrophilic cleft of the B fragment(s), left over in themembrane (Miesler, 1983; Montecucco et al., 1985), islikely to reduce its size to minimize interactions with thehydrocarbon chains of lipids. However, the presence of atransmembrane alignment of hydrophilic residues is expectedto give rise to an ion channel. Such a feature would accountfor the low conductance of DT ion channels better than apolypeptide B pore, which, having to accommodate the sizeof a polypeptide chain (though in an extended form), isexpected to show a conductance similar to or higher thanthat of pore-forming proteins (Colombini, 1980; Menestrina,1986).Clearly more experiments are needed to clarify the

mechanism of protein translocation across membranes, butin the case of DT we are beginning to unravel the molecularsteps involved in the process.

Materials and methodsDT and its mutant forms crm 45, crm 1001 and crm 197 were preparedfrom culture filtrates of the appropriate C.diphtheriae strains as describedby Rappuoli et al. (1983). The toxins were nicked by TPCK-treated trypsin3358

(Serva) and checked by SDS-PAGE. 95-98% of DT was present in di-chain form in different experiments and no more than 5% degraded materialwas present in nicked crm 45, crm 197 and crm 1001. Toxins were storedat -80°C in 10 mM NaPi, 125 mM NaCI, pH 7.4. Amiloride, ouabainand gramicidin D were from Sigma, 3,3'-Dipropylthiadicarbocyanine iodide[diS-C3-(5)] was a gift from Dr Alan Waggoner (Amherst College, MA).L-[3U-_4C]Leucine (sp. act. 342 Ci/mol) was purchased from AmershamInternational (Amersham, UK). Eagle's minimum essential medium (MEM)and fetal calf serum (FCS) were from Flow.

Cell culturesVero cells (from African green monkey kidney) and CHO cells (from Chinesehamster ovary) were grown as monolayers at 37°C in plastic flasks in Eagle'sMEM supplemented with 10% fetal calf serum. Balb/c mice and guinea-pig spleen lymphocytes were isolated from the tissue omogenate by stepgradient centrifugation on 6% Ficoll-Hypaque (Pharmacia) as describedpreviously (Rink et al., 1980).

Protein synthesis inhibitionVero and CHO cells were seeded into 24-well disposable trays the day beforethe experiments at a density of 105/well. Cells were incubated for 1 h at4°C with toxin in MEM containing 10% FCS, 10 mM Na-Hepes, 10 mMNH4Cl, pH 7.4. After washing twice with the same cold medium withoutNa-Hepes, medium A (123 mM NaCl, 6 mM KCI, 0.8 mM MgCl2,1.5 mM CaCI2, 5 mM NaPi, 5 mM citric acid, 5.6 mM glucose, 10 mMNH4Cl) adjusted to the desired pH with 8% phosphoric acid andprewarmed at 37°C was added and the incubation was prolonged for 5 min.The medium was removed and the cells were washed twice with MEMand further incubated with MEM containing 10 mM NH4Cl for 2 h at37°C. In some experiments the intoxication with DT4 was preceded by a10-min incubation with 100 /AM ouabain at 37°C followed by a cold washwithout ouabain and then cells were treated as above with the exceptionthat ouabain was present during the low pH pulse.

In another set of experiments an alternative procedure was followed: 0.8 mlof medium A, pH 7.4 containing different concentrations of toxin was addedat 37°C and the pH was immediately lowered to the desired value by adding0.2 ml of acidic medium A; 5 min later cells were washed and furtherincubated as described above. The effect of DT on protein synthesis wasmeasured as radioactivity incorporated during a 15-min pulse with a MEMmedium containing 50 nCi/ml of [14C]leucine and no cold leucine.The number of A chains per cell was estimated following the assumptions

of Chung and Collier (1977) with a kinetic constant of 0.83 x 10-9 M-lmin-I at 37°C (Moynihan and Pappenheimer, 1981) and a cell volumeof 2 x 10-121.

K+ and Na+ measurement by flame photometryAfter the above-described low pH incubation, cells were washed with coldcholine medium (129 mM choline-Cl, 0.8 mM MgCl2, 1.5 mM CaC12,5 mM H3P04, 5 mM citric acid, 5.6 mM glucose adjusted to pH 7.4 withTris -OH) containing 10 mM NH4CI and then dissolved with 100 td/wellof 0.5% (w/v) Triton X-100. Ten minutes later the cell lysate was recovered,diluted in 1 ml of bidistilled water and its K+ and Na+ content measuredby flame photometry with a Perkin-Elmer 305 B atomic absorptionphotometer.

Determination of DT-induced K+ release from cells with aK + -selective electrodeVero cells, grown to confluence in 50 ml culture plastic flasks (Falcon),were incubated for 15 min at 25°C with various concentrations of DT inMEM, pH 7.4, and then washed twice with choline medium, pH 7.4. Flaskswere filled with choline medium (to avoid interference with the electrode)previously acidified to the desired value with phosphoric acid. The potassium-selective electrode (Schott, Mainz, FRG) was immediately immersed in thesolution and the signal recorded. At the end of the experiment, 4 ytg/mlof gramicidin D was added. Absolute quantitation of K+ was obtained bytitration with a KCI standard solution under the same conditions.

Measurement of membrane potentialTrypsin-treated Vero and CHO cells or lymphocytes were washed twiceby centrifugation and resuspended in medium A at a density varying between1 and 8 x 106/ml. 0.5 x 106 Vero or CHO cells or 4 x 106 lymphocytesin 2 ml of medium A were placed in a thermostatted and stirred cuvetteof a Perkin Elmer 650-40 spectrophotometer at 37°C. diS-C3-(5) from a100 tiM stock solution in distilled DMSO was added to a final concentrationof 200 nM. When the fluorescence signal (excitation: 620 nm, emission:660 nm, slits 10 nm) was stabilized, DT was added and 1 -5 minlater thesolution was acidified with 8% phosphoric acid. The change in fluorescencedue to the pH variation was subtracted and the value obtained was expressed

Membrane permeability changes induced by diphtheria toxin at acidic pH

as a percentage of the value obtained with 4 Ag/ml gramicidin D in the sameconditions.

Acknowledgements

We thank Drs P.Boquet, G.Menestrina and T.Pozzan for critical readingof the manuscript and Professor G.F.Azzone for encouragement and support.We are indebted to Dr S.Harshman (Vanderbilt University, Nashville) forthe gift of a sample of purified S.aureus a-toxin. The present research waspartially supported by a grant from the Regione Veneto. This work is inpartial fulfilment of the doctorate degree of the University of Padova in'Molecular and Cellular Biology and Pathology' of E.P.

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