On the membrane translocation of diphtheria toxin: at ... - Europe PMC

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

On the membrane translocation of diphtheria toxin: at low 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 'Centro Ricerche SCLAVO S.p.A., Via Fiorentina 1, 53100 Siena, Italy Communicated by G.Cesareni

Diphtheria toxin (DT) in acidic media forms ionconducting channels across the plasma membrane and inhibits protein synthesis of both highly and poorly DTsensitive cell lines. This results in loss of cell potassium and in entry of both sodium and protons with a concomitant rapid lowering of membrane potential. The pH dependency of the permeability changes is similar to that of the inhibition of cell protein synthesis. DT-induced ion channels close when the pH of the external medium is returned to neutrality and cells recover their normal monovalent cation content. Similar permeability changes were induced by two DT mutants defective either in enzymatic activity or in cell binding, but not with a mutant defective in membrane translocation. The implication of these findings for the mechanism of DT membrane translocation is discussed. Key words: diphtheria toxin/ion channels/monovalent cations/membrane potential

Introduction Diphtheria toxin (DT) is a very powerful protein toxin responsible for clinical diphtheria (Pappenheimer, 1981). It is produced by Corynephago (tox+) infected Corynebacterium 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 proteases into two fragments connected via a disulphide bridge: the A 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). Their mechanism of cell intoxication can be conveniently divided into three main steps: (i) cell binding, (ii) membrane translocation and (iii) cytoplasmic target modification. There is evidence that both a plasma membrane protein(s) and phospholipids are involved in the binding of DT to cells (Moehring and Crispell, 1974; Alving et al., 1980; Olsnes et al., 1985; Cieplak et al., 1987; Papini et al., 1987a). The third step of DT action involves the ADP-ribosylation, catalysed by fragment A, of elongation factor 2 with consequent 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 the hydrophobic membrane barrier. Available evidence indicate that DT enters the cytoplasm from a low pH compartment (Olsnes and Sandvig, 1985). Experiments performed with a variety of techniques have shown that DT undergoes a lowpH-driven conformational change that occurs in a range of acidic pHs overlapping that found in endosomes (Sandvig and Olsnes, 1981; Blewitt et al., 1985; Montecucco et al., 1985; Papini et al., 1987b,c). This structural change results in the exposure of hydrophobic surfaces that enable the toxin to enter in contact with the hydrocarbon chains of phospholipids 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 with this model system, a mechanism has been suggested for the entry of DT into cells. It was proposed that at acidic pHs fragment B forms a transmembrane tunnel large enough to accommodate the A chain in an extended form. The hydrophilic A chain unfolds at low pH and transverses the membrane inside the tunnel, shielded from the contact with the hydrocarbon tails of lipids (Kagan et al., 1981; Hoch et al., 1985). Even though this tunnel model does not accommodate some recent observations, it offers an interesting hypothesis for the little-understood process of protein membrane translocation: that it is not limited to toxins, but occurs in cells for all those proteins imported from 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 the process of cell intoxication. Cells were treated with DT at acidic pHs in order to introduce the toxin from the plasma membrane (Sandvig and Olsnes, 1980, 1981; Draper and Simon, 1980). The effects of DT on plasma membrane permeability and on protein synthesis of both highly and poorly DT-sensitive cells were investigated and compared with those caused by some DT mutants that cross-react immunologically with DT but are defective either in cell binding (crm 45) or in membrane translocation (crm 1001) or in enzymic activity (crm 197).

Results Modifications of K+ and Na+ cellular contents induced by DT Studies with model systems have shown that at acidic pHs DT forms ion channels across lipid bilayers (Donovan et al., 1981; Kagan et al., 1981; Miesler, 1983; Zalman and Wisnieski, 1984; Hoch et al., 1985). Here we have tested the possibility that DT ion channels also assemble on the plasma membrane of living cells during cell intoxication by

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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 described in Materials and methods. Na+ (E-El) and K+ (0-0) cellular contents were determined by flame photometry after a 15-min incubation with DT at 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 is nearly maximal and prolonged exposure to low pH leads to a decrease in the amount of leucine incorporated by controls. On the other hand, K+ and Na+ were measured after 15 min to have a better chance to trace their fluxes. As the control value of intracellular Na+ concentration is difficult to estimate 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 experiments run in triplicate and bars represent standard deviations.

measuring the K+ and Na+ content of cells after incubation with the toxin at low pH. If pores are assembled on the plasma membrane, potassium ions should leak out while sodium ions should enter into the cell. Figure IA shows that the K+ and Na+ content of Vero cells, incubated for 1 h at 4°C with DT, washed and then exposed to pH 4.9 for 15 min at 37°C, changes as a function of DT concentration. In the presence of 10-7 M DT, the intracellular concentration of potassium is reduced from 140 mM (Arosen, 1985) to -40 mM while that of sodium increases from the basal value of 9 mM (Arosen, 1985) to 125 mM; low pH alone has no effect (not shown). Na+ influx and K+ efflux are already detectable at toxin concentrations as low as 6 x 10-10 M and the ratio between the two fluxes is always > 1, indicating that sodium uptake is more pronounced than potassium efflux. Figure lA also shows that the dose-response curve of the inhibition of protein synthesis, measured under the same conditions, is shifted by at least one log unit toward lower toxin concentrations as compared to those relating to ion fluxes; with 10-10 M DT, protein synthesis is 90% inhibited while the effect on K+ and Na+ levels begins to be detectable. Ouabain does not modify these patterns (not shown) indicating that the shift between the cytotoxic and permeability effects is not due to a compensatory activation of the Na+/K+-ATPase. The above-described results were obtained with Vero cells, a line highly sensitive to DT with a number of high-affinity DT receptors estimated to be 105/cell (Middlebrook et al., 1978; Mekada et al., 1982). However DT ion channels were observed in lipid model systems lacking such receptors. Hence investigations were extended to CHO cells that are poorly sensitive to DT because they possess 100-fold less high-affinity receptors per cell (Mekada -

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

et al., 1982). Figure lB shows that also under the present conditions CHO cells are less sensitive than Vero cells with a protein synthesis inhibition curve shifted toward much higher DT concentrations. The shift is closely related to the difference in the number of DT receptors between the two cells lines. None the less, even in this case, DT-induced ion

Membrane permeability changes induced by diphtheria toxin at acidic pH

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DT (- log M) Fig. 3. Permeability and cytotoxic effects induced in CHO cells by crm 45 and crm 1001. Protein synthesis inhibition (A) and monovalent ), crm 45 (------) and crm 1001 (. ) measured as described in the legend to Figure 2. Points are fluxes (B) induced by DT ( two experiments run in triplicate; standard deviations are shown as bars.

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channels are present and again there is a shift between the dose dependency curves of protein synthesis inhibition and the 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, unbound toxin is present in the medium at the time of acidification. The two situations may be different since it is known that DT aggregates in acidic solutions and such DT aggregates could be responsible, upon partition into the lipid bilayer, for the formation of ion-conducting channels. Hence we have tested the effects of DT on cells under conditions similar to those used with artificial membranes. DT was added in a physiological buffer at pH 7.4 to CHO cells at 37°C in the presence of 10 mM NH4Cl, to inhibit DT entrance in the cytosol from intracellular acidic compartments, and the pH of the medium was lowered by adding an aliquot of acidic buffer. Figure 2 shows that under these conditions there is an increase in protein synthesis inhibition (- 1 log unit) accompanied by a much larger effect on the permeability to monovalent cations ( 2 log units). A similar finding was obtained 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 protein synthesis indicates that, under such conditions, DT can be assembled into forms that are more efficient in increasing membrane permeability than in translocating fragment A. Figure 2 also shows that the presence of amiloride, an inhibitor of Na+/H+ and Na+/Ca2+ antiporters (Smith et al., 1982; Aronsen, 1985), does not affect K+ and Na+ fluxes, thus suggesting that Na+ influx is not due to activation of such sodium antiporters. Effect of DT mutants monovalent cations

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Crm 45 lacks a 12-kd C-terminal segment of the B fragment (Giannini et al., 1984), binds to cells with low affinity

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(Boquet and Pappenheimer, 1976), forms channels across planar lipid bilayers (Kagan et al., 1981) and inserts into lipid 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 synthesis 100-fold was inhibited in a range of toxin concentrations higher than with DT. As with DT, changes in the cell contents of K+ and Na+ are detectable only at toxin concentrations where protein synthesis is nearly completely inhibited. Crm 1001 has a single amino acid substitution (Cys-471 replaced by Tyr) (G.Ratti et al., unpublished results), binds to cells as well as DT does and has a similar enzymatic activity (Zucker, 1983). However, it has a very low toxicitiy because it is defective in membrane translocation (Papini et al., 1987c). Also, under the conditions used here crm 1001 is poorly toxic (Figure 3). Moreover, at concentrations as high as 10-7 M it does not induce any change in the permeability of CHO cells to monovalent cations. Hence there appears to be a correlation between the ability of DT to translocate its A fragment in the cytoplasm and that of inducing the formation of ion channels. Moreover, this result is consistent with the idea that the DT-induced changes of permeability are due to channels formed by the toxin and not 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 pHs as well as DT does (Papini et al., 1987a). Crm 197 alters the permeability of Vero cells to monovalent cations in a manner comparable to that of DT but without any effect on protein synthesis (not shown). This experiment shows that the inhibition of protein synthesis found above is not to be attributed to the alteration of permeability brought about by DT 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 of endosomes. -

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pH Fig. 4. pH dependence and reversibility of DT-induced changes in K+ and Na+ cellular contents of CHO cells. Panel A shows the pH dependence of 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). Panel B 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 amount of 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 then further incubated for the periods indicated at 37°C in MEM medium, pH 7.4. Symbols as in Figure 1; standard deviation bars are omitted for

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Taken together, the results obtained with DT mutants indicate that the change in permeability is caused by the N-terminal 25-kd part of fragment B in agreement with the results obtained with planar lipid bilayers (Kagan et al., 1981; Miesler, 1983). pH and time dependence and reversibility of DTinduced cellular modifications To be relevant to the process of DT membrane translocation, the pH dependence of the DT-induced change of permeability should fall within the range of pHs found in endosomes (Mellman et al., 1986). Figure 4 A and B shows that the cellular effects of DT are maximal already at pH 5 and that above pH 5 the effects of DT on protein synthesis and on the cellular content of sodium and potassium have a similar pH dependence. Figure 4C also shows that the inhibition of protein synthesis is not due to a membrane lytic effect of DT because the normal cellular content of sodium and potassium can be regained by cells after a short recovery

period. As found above, the two phenomena induced by DT are not comparable because the permeability to both K+ and to Na+ rapidly decreases below pH 5, while the inhibition of protein synthesis remains at the same level. Such a bellshaped pH dependence of permeability could reflect the existence, at pHs lower than 5.0, of a toxin state less effective in inducing cation transmembrane fluxes although still able to transfer the A chain into the cytosol; or could result from a conversion of DT channels from a cation to an anion selectivity. Such conversion has been shown to occur on planar 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 content starts seconds after acidification and continues for at least

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

15 min (Figure 4A); more prolonged exposures to low pH could not be tested because these start to cause irreversible cell damage. Taken together, the present findings are in agreement with the idea that at low pH DT forms across the plasma membrane ion-conducting channels that remain open as long as a transmembrane pH gradient is present; when external pH is returned to neutrality, channels close and the cell rapidly re-establishes a normal ion content before any effect consequent to inhibition of its protein synthesis can be noticed.

Membrane permeability changes induced by diphtheria toxin at acidic pH

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cell uptake. Acidification causes a fluorescence increase due to a direct effect on dye fluorescence intensity and to a depolarizing effect on protons (Sandvig et al., 1986). Despite these effects, that lower the sensitivity of this assay, it clearly appears that DT at low pH is able to depolarize fibroblasts and lymphocytes further. Again its effect appears to be rather slow since gramicidin added after DT caused a further increase in fluorescence. For a comparison, Figure 6 also reports the effect on membrane potential of the a-toxin of Staphylococcus aureus, which causes haemolysis and forms large pores across the plasma membrane (Bhakdi et al., 1981; Menestrina, 1986).

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Fig. 6. Low pH DT-induced depolarization of cells detected with a fluorescent probe. Traces show the change of fluorescence of the membrane potential probe diS-C3(5) after uptake by Vero cells and mouse spleen lymphocytes following acidification of the medium to pH 4.9 in the presence of different concentrations of DT (reported below each curve with 8% phosphoric acid. Gram indicates the addition of gramicidin D (4 jig/ml) and a-toxin the addition of S.aureus (x-toxin (0.5 ztM final concentration). Fluorescence is reported in arbitrary units.

DT-induced K+ release measured with a potassiumselective electrode The effiux of intracellular potassium caused by DT at acidic pH was confirmed by following its appearance in the extracellular medium. We have tested both spleen lymphocytes resuspended in choline medium and CHO or Vero cells adherent to the wall of a plastic bottle. Figure 5 shows the record from a K+-sensitive electrode immersed in the medium bathing a monolayer of Vero cells covering one wall of the flask. The kinetics and pH dependence (not shown) of the K+ release induced by DT are very similar to those obtained by measuring cellular ion contents by flame photometry. The absence of calcium (panel B) does not modify the K+ efflux and, since no sodium ions were present in the medium, it is very unlikely that potassium is released from cells in conjunction with sodium or calcium influxes. On the other hand, the possibility that K+ efflux is due, at least in part, to a proton influx that activates a DT-independent K+ efflux pathway cannot be excluded. Similar results were obtained with CHO cells and with mouse

spleen lymphocytes (not shown). DT at acidic pHs lowers membrane potential The fluorescent probe diS-C3(5) has been extensively used to follow changes in membrane potential (Waggoner, 1976; Tsien and Hladky, 1978; Rink et al., 1980). This merocyanine dye is taken up by cells in a membrane-potentialdependent process and the binding to cell structures leads to a quenching of its fluorescence. Depolarization causes dye release and a consequent recovery of fluorescence. Figure 6 shows the decrease in dye fluorescence accompanying its

Discussion Membrane translocation is the least understood step in the process of cell intoxication by toxins with intracellular targets; somehow a water-soluble molecule such as DT becomes able to cross the hydrophobic membrane barrier. This passage is common to a variety of proteins that are synthesized in the cytoplasm and are to be localized in inner compartments of cell organelles such as endoplasmic reticulum, mitochondria and chloroplasts (Zimmermann and Meyer, 1986; Eilers and Schatz, 1988). The first reports bearing on the membrane translocation of DT showed that DT causes a pH-dependent increase in the conductance of planar 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 cations of cells. The simplest interpretation of these results is that DT at low pH forms ion-conducting channels in living cells, resulting in potassium efflux and sodium and proton influx with a consequent decrease of membrane potential. This is supported by the finding that crm 1001 binds to cells but does not cause any change in permeability. However, we cannot exclude the possibility that at least part of the fluxes occur via a DT-independent mechanism activated by acidification. It has been suggested that at low pH fragment B forms a transmembrane tunnel that allows the passage of the enzymatic fragment A in an extended form (Kagan et al., 1981; Hoch et al., 1985). In this model the formation of a large ion-conducting pore is inherent to the process of fragment A entry into the cell and hence the two phenomena should be related. Here we present evidence that there is a range of DT concentrations that cause a large inhibition of protein synthesis without a measurable effect on the cellular content of monovalent cations; when 90% or more of Vero and CHO cells protein synthesis was inhibited, 20 A fragments had penetrated into the cytoplasm during the 5 min of low pH incubation with a very small, or non-measurable, effect on cell sodium even after 15 min. Moreover, the DT-induced change in cell potassium and sodium is rather slow, and this is unexpected on the basis of the conductance measured in planar lipid bilayers. Assuming that DT forms pores of similar conductance in model and cellular systems, one can estimate that sodium ions should have equilibrated across the plasma membrane in 15 min. This discrepancy between artificial and cellular systems cannot be ascribed to DT receptors because similar results were obtained with -

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cells differing in their number of toxin receptors and also with crm 45 which is unable to bind to cells with high affinity. Indeed recent evidence indicates that DT receptors are needed for driving the toxin into endosomal compartments, but not for its translocation into the cytoplasm (Colombatti et al., 1986; Greenfield et al., 1987; Murphy et al., 1986; Bacha et al., 1988). The simplest explanation for the difference between cellular and model systems is that DT ion channels assembled at low pH on cells have either a lower mean conductance or are closed for most of their life-time. Some results have been recently reported that cannot be easily accommodated in a protein tunnel model. Sandvig et al. (1986) have shown that the reduction of cell membrane potential does not lower the ability of DT to translocate its enzymic A part in the cytosol, while DT ion channels across planar lipid bilayers are voltage dependent (Donovan et al., 1981; Kagan et al., 1981). Moreover, hydrophobic photolabelling experiments have shown that at low pH both fragment B and fragment A interact with the fatty acid chains of phospholipids (Hu and Holmes, 1984; Zalman and Wisnieski, 1984; Montecucco et al., 1985; Papini et al., 1987c) and that the pH dependence of this phenomenon is closely related to that of DT intoxication of cells subjected to 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 (Bisson and Montecucco, 1987; Papini et al., 1987c; Singer et al., 1987). In this model it is envisaged that, at low pH, a B fragment(s) inserts in the lipid bilayer, with its hydrophobic surfaces exposed to lipids, and forms a hydrophilic cleft that faces the hydrophilic residues of an A fragment. At variance with the tunnel model, the A fragment remains in contact with lipids via its hydrophobic residues during its membrane translocation. The matching of hydrophilic and hydrophobic types of interactions between lipids, A chain and B chain would reduce the energetic cost of the process as compared to the tunnel model where the hydrophilic B channel has to accommodate not only the hydrophilic residues of the A chain but also the 68 hydrophobic residues present in its 193-residue-long sequence. After refolding and release of the A chain in the cytoplasm by disulphide reduction, the hydrophilic cleft of the B fragment(s), left over in the membrane (Miesler, 1983; Montecucco et al., 1985), is likely to reduce its size to minimize interactions with the hydrocarbon chains of lipids. However, the presence of a transmembrane alignment of hydrophilic residues is expected to give rise to an ion channel. Such a feature would account for the low conductance of DT ion channels better than a polypeptide B pore, which, having to accommodate the size of a polypeptide chain (though in an extended form), is expected to show a conductance similar to or higher than that of pore-forming proteins (Colombini, 1980; Menestrina, 1986). Clearly more experiments are needed to clarify the mechanism of protein translocation across membranes, but in the case of DT we are beginning to unravel the molecular steps involved in the process.

Materials and methods DT and its mutant forms crm 45, crm 1001 and crm 197 were prepared from culture filtrates of the appropriate C.diphtheriae strains as described by Rappuoli et al. (1983). The toxins were nicked by TPCK-treated trypsin

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(Serva) and checked by SDS-PAGE. 95-98% of DT was present in dichain form in different experiments and no more than 5% degraded material was present in nicked crm 45, crm 197 and crm 1001. Toxins were stored at -80°C in 10 mM NaPi, 125 mM NaCI, pH 7.4. Amiloride, ouabain and 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 Amersham International (Amersham, UK). Eagle's minimum essential medium (MEM) and fetal calf serum (FCS) were from Flow.

Cell cultures Vero cells (from African green monkey kidney) and CHO cells (from Chinese hamster ovary) were grown as monolayers at 37°C in plastic flasks in Eagle's MEM supplemented with 10% fetal calf serum. Balb/c mice and guineapig spleen lymphocytes were isolated from the tissue omogenate by step gradient centrifugation on 6% Ficoll-Hypaque (Pharmacia) as described previously (Rink et al., 1980).

Protein synthesis inhibition Vero and CHO cells were seeded into 24-well disposable trays the day before the experiments at a density of 105/well. Cells were incubated for 1 h at 4°C with toxin in MEM containing 10% FCS, 10 mM Na-Hepes, 10 mM NH4Cl, pH 7.4. After washing twice with the same cold medium without Na-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 mM NH4Cl) adjusted to the desired pH with 8% phosphoric acid and prewarmed at 37°C was added and the incubation was prolonged for 5 min. The medium was removed and the cells were washed twice with MEM and further incubated with MEM containing 10 mM NH4Cl for 2 h at 37°C. In some experiments the intoxication with DT4 was preceded by a 10-min incubation with 100 /AM ouabain at 37°C followed by a cold wash without ouabain and then cells were treated as above with the exception that ouabain was present during the low pH pulse. In another set of experiments an alternative procedure was followed: 0.8 ml of medium A, pH 7.4 containing different concentrations of toxin was added at 37°C and the pH was immediately lowered to the desired value by adding 0.2 ml of acidic medium A; 5 min later cells were washed and further incubated as described above. The effect of DT on protein synthesis was measured as radioactivity incorporated during a 15-min pulse with a MEM medium 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-l min-I at 37°C (Moynihan and Pappenheimer, 1981) and a cell volume of 2 x 10-121. K+ and Na+ measurement by flame photometry After the above-described low pH incubation, cells were washed with cold choline 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 with Tris -OH) containing 10 mM NH4CI and then dissolved with 100 td/well of 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 measured by flame photometry with a Perkin-Elmer 305 B atomic absorption photometer.

Determination of DT-induced K+ release from cells with a K + -selective electrode Vero cells, grown to confluence in 50 ml culture plastic flasks (Falcon), were incubated for 15 min at 25°C with various concentrations of DT in MEM, pH 7.4, and then washed twice with choline medium, pH 7.4. Flasks were filled with choline medium (to avoid interference with the electrode) previously acidified to the desired value with phosphoric acid. The potassiumselective electrode (Schott, Mainz, FRG) was immediately immersed in the solution and the signal recorded. At the end of the experiment, 4 ytg/ml of gramicidin D was added. Absolute quantitation of K+ was obtained by titration with a KCI standard solution under the same conditions. Measurement of membrane potential Trypsin-treated Vero and CHO cells or lymphocytes were washed twice by centrifugation and resuspended in medium A at a density varying between 1 and 8 x 106/ml. 0.5 x 106 Vero or CHO cells or 4 x 106 lymphocytes in 2 ml of medium A were placed in a thermostatted and stirred cuvette of a Perkin Elmer 650-40 spectrophotometer at 37°C. diS-C3-(5) from a 100 tiM stock solution in distilled DMSO was added to a final concentration of 200 nM. When the fluorescence signal (excitation: 620 nm, emission: 660 nm, slits 10 nm) was stabilized, DT was added and 1 -5 minlater the solution was acidified with 8 % phosphoric acid. The change in fluorescence due 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 conditions.

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gramicidin D in the same

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

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