Role of endogenous channels in red blood cells response to their exposure to the pore forming toxin Sticholysin II

Toxicon 46 (2005) 297–307 www.elsevier.com/locate/toxicon

Role of endogenous channels in red blood cells response to their exposure to the pore forming toxin Sticholysin II G. Celedona, F. Venegasa, A.M. Camposb, M.E. Lanioc, D. Martinezc, C. Sotoc, C. Alvarezc, E. Lissid,* a

Facultad de Ciencias, Universidad de Valparaı´so, Valparaı´so, Chile Instituto de Quı´mica, Universidad Cato´lica de Valparaı´so, Valparaı´so, Chile c Facultad de Biologı´a, Universidad de La Habana, Havana, Cuba d Departamento de Quı´mica, Facultad de Quı´mica y Biologı´a, Universidad de Santiago de Chile, Casilla 40-Correo 33, Santiago, Chile b

Received 27 August 2004; accepted 15 April 2005 Available online 29 June 2005

Abstract Sticholysin II (St II) is a highly hemolytic cytolysin isolated from the sea anemone Stichodactyla heliantus. The toxin hemolytic action takes place through the formation of channels that provoke an electrolyte unbalance leading to osmotic shock. The lytic event must involve the exchange of electrolytes and the entrance of water, leading to red blood cell disruption. These processes can occur through St II pores and/or the endogenous red blood cells transporters. In order to evaluate the contribution of these channels to water, anion and cation transport, we have measured the hemolysis and KC efflux rates in the presence of several specific inhibitors. The results obtained in the presence of Hg, an AQP1 blocker, indicate that water transport through these channels is not essential for the occurrence of the lytic process induced by St II. The data also support a partial role of KC and anion transporters. In particular, they are compatible with a preferential KC efflux though the KC/ClK co-transport as a response to the promoted swelling. Furthermore, they suggest that chloride influx, a process that can regulate both KC efflux and lysis, is partially mediated by the endogenous cell transporters, in particular, band-3 anion exchange system being relevant at early stages of the lytic process. q 2005 Elsevier Ltd. All rights reserved. Keywords: Sticholysin II; Hemolytic activity; KC ion efflux; Cation transporters; Band-3

1. Introduction St II is a basic cytolysin produced by the sea anemone Stichodactyla heliantus, that belongs to the actinoporin protein family, characterized by high pI, molecular size around 20 kDa (Lanio et al., 2001), inhibition by sphingomyelin (Alvarez-Valcarcel et al., 2001), and predominant bstructure (Manchen˜o et al., 2003; Martı´nez et al., 2001; * Corresponding author. Departamento de Quı´mica, Universidad de Santiago de Chile, Casilla 40-Correo 33, Santiago, Chile. Tel.: C56 2 68 12 108. E-mail address: [email protected] (E. Lissi).

0041-0101/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2005.04.017

Menestrina et al., 1999). St II forms cation selective oligomeric pores in model lipid membranes (Tejuca et al., 1996, 2001). The hemolytic activity (HA) of these poreforming toxins takes place with a kinetics similar to that of the osmotic shock promoted by suspending the red blood cells (RBC) in hypotonic media. Under the proper conditions, the kinetics of the hemolytic process presents a lag phase, followed by a very fast disruption of the cell ensemble (Campos et al., 1999; Pazos et al., 1998). However, there are basic differences between the hypotonic shock and the lytic process elicited by these toxins. The hyposmotic shock involves water and ion transport through endogenous mechanisms operating in the red blood cells:

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passive water transport through the membrane and/or water channels (aquaporins) and ion transport through the numerous channels that are normally operating in the cell and/or that can be activated by the swelling process (Guizouarn and Motais, 1999). On the other hand, the HA of cytolysins is due to the formation of pores that can promote a net influx of ions that ultimately leads to the RBC swelling and disruption. In these systems, water, anions (i.e. chloride) and cations (such as NaC and KC and other cations possibly present, such as Ca2C) could be transported by the RBC endogenous mechanisms and/or through the toxin pores (Guizouarn and Motais, 1999; Macek et al., 1994; Tejuca et al., 1996). The lytic process is then more complex, and the role of endogenous channels cannot be a priori assumed or disregarded. In order to contribute to the understanding of this matter, we have performed studies of HA and KC efflux promoted by Sticholysin II (St II) under a variety of conditions aimed to establish the role of endogenous RBC channels relevant to the flux of water, anions (chloride) and cations (KC). The basic rationale of our experimental design is that the lytic process must involve a net influx of cations (NaC) and water, that KC efflux precedes the lytic process (Macek et al., 1994; Martı´nez et al., 2001), that this KC efflux can be a RBC defense mechanism (Lang et al., 1998; 2003), and that KC efflux and NaC influx in cell osmotic lysis is generally associated to chloride co-transport (Lang et al., 1998; Motais et al., 1991).

2. Materials and methods Toxins and reagents. St II was purified according to Lanio et al. (2001). Reagents employed were from Sigma or Aldrich. Polyethylene glycol (PEG 6000) was obtained from Fluka.

2.2. Potassium ions efflux Potassium release from the RBC was measured with an ion selective electrode (Cole Palmer 27502-38, 39, USA) coupled to an Orion 420A millivoltmeter. Lineal relationships were obtained between the measured voltage and the logarithm of KC concentration in calibrations employing KCl in saline buffer solution. Calibrations were carried out in the presence of the specific inhibitors. The slope of the plot was that expected from a Nerst type relationship. The raise of KC concentration after exposing the RBC suspension to the toxin was followed during ca. 20 min under constant stirring. Total release was estimated by addition of a large excess of toxin. 2.3. Osmotic fragility RBC suspensions, pretreated with Hg2C, were added to solutions containing increasing NaCl concentrations. After 60 min incubation at room temperature, the solutions were centrifuged. The percentage of hemolysis was determined spectrophotometrically at 540 nm (Sztiller and Puchala, 2003). Control experiments were carried out without previous incubation with Hg2C. 2.4. Blockage of KC transport This was achieved by pre-incubation of the RBC suspension with 10 mM ouabain (NaC/KC pump; Lauf et al., 1985), 10 mM bumetanide (NaC/KC/2 ClK cotransport; Lauf et al., 1985), 10 mM nitrendipine (Ca2C activated KC channel: Gardos; Chandy et al., 2001; Ellory et al., 1992) or 2 mM furosemide (KC/ClK co-transport; Bursell and Kirk, 1996; Kaji, 1986). Blockage (inespecific) of KC channels was achieved by measuring the HA and K ion efflux in presence of BaCl2 (up to 400 mM; Jiang and MacKinnon, 2000).

2.1. Hemolytic activity assays Hemolysis was assayed by measuring the decrease in turbidity of a human RBC suspension at 600 nm in a microplate reader (Multiscan) (Pederzolli et al., 1995) or in a 3 mL cuvette with 1 cm length path (Campos et al., 1999). Erythrocyte suspensions were prepared using human RBC from volunteer donors, washed and re-suspended in physiological buffer solutions (TBS: 0.145 M NaCl, 10 mM Tris–HCl, pH 7.4 or PBS: 0.145 M NaCl, NaH2PO4 1.05 mM, NaHPO4 3.95 mM, pH 7.4). The concentration of the standard RBC suspension was adjusted to obtain absorbances of 1.0 and 0.1 in the 3 mL cells or in the microplate reader wells, respectively. This corresponds to approximately 5.2!107 or 4.1!107 cells/mL. Total hemolysis was estimated by adding an excess of the toxin. All experiments were carried out at 22G1 8C.

2.5. Blockage of the band-3 anion transport To block this transport, the RBC suspension was preincubated during 30 min at 25 8C with 50 mM 4,4 0 diisothiocyanatostilbene-2,2 0 -disulfonic acid (DIDS) or 100 mM 4,4 0 -dinitrostilbene-2,2 0 -disulfonic acid (DNDS) (Cabantchik and Greger, 1992). 2.6. Blockage of the RBC aquoporin1 (AQP1) Water transport through AQP1 was inhibited by carrying out the assays in the presence of 50 mM HgCl2, concentration able to provoke the inhibition of this channel as reported by Tsai et al. (1991). The suspension was preincubated during 10 min in the presence of the Hg2C ions.

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2.7. Replacement of the external NaC by KC ions The RBC suspension was prepared employing KC instead of NaC in PBS. NaC/KC equimolecular conditions were obtained by mixing equal volumes of RBC suspensions prepared employing sodium or potassium salts. 2.8. Replacement of the external chloride by nitrate Sodium nitrate was employed to partially replace sodium chloride in PBS. To allow the exchange of the internal anions, the red blood suspension was incubated in the presence of the nitrate rich buffer containing 45 mM sodium nitrate plus 95 mM NaCl during 30 min at 37 8C. 2.9. Modulation of the free Ca2C concentration Double distilled water employed in the present experiments contained up to 10 mM Ca ions, as detected by atomic absorption spectroscopy. In some experiments, the free Ca2C concentration was regulated according to Durham (1983). Free concentrations of Ca2C of 0.6 mM (pCaZ3.2) was obtained employing Hepes buffer, pH 7.4 (10 mM sodium citrate; 9.75 mM CaCl2; 19.94 mM Na-Hepes; 20.06 mM Hepes; 90 mM NaCl). This buffer (300 mOsm) was employed in the preparation of the standard RBC suspension. 2.10. Modification of the medium osmolality by a non-permeant solute PEG 6000 was employed as a solute unable to permeate through the toxin pores (Tejuca et al., 2001). The standard RBC suspension prepared in PBS was incubated with 10 mM PEG 6000 during 5 min before performing the HA and KC efflux assays. 2.11. Vesicle permeabilization experiments The rate and extent of bilayers permeabilization by St II were evaluated by following carboxyfluorescein release from egg phosphatidylcholine:sphingomyelin (PC:SM, 1:1) large unilamellar vesicles (LUVs) (Dalla Serra and Menestrina, 2003; Tejuca et al., 1996). Vesicles were prepared by the extrusion method (McDonald, 1991) submitting multilamellar vesicles (MLVs) to a freezing/ thawing cycle and 31 extrusion cycles (27 8C) through polycarbonate filters with a pore size of 100 nM (Nucleopore, USA). Vesicles were prepared in the presence of carboxyfluorescein (80 mM, pH 7.5). Non-encapsulated probe was separated from the vesicle suspension through a Sephadex G-75 gel filtration column. Lipid concentration, determined according to Fiske and Subarrow (1925), was 2 mM in the permeabilization assays. All procedures were carried out in buffer solutions comprising Tris–HCl 20 mM, NaCl 140 mM, pH 7.0. Vesicle permeabilization following

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St II incorporation was evaluated from the increase in fluorescence intensity associated to the carboxyfluorescein release. Fluorescence emission was measured at 520 nm after excitation at 490 nm in a spectrofluorometer Shimadzu (Japan) using a 1-cm path quartz cuvette containing 3 mL of buffer. Spontaneous leakage of carboxyfluorescein was negligible under these conditions. Maximum release (Fmax) was obtained by adding Triton X-100 (0.1%, final concentration). The percentage of release was calculated as: f ð%Þ Z 100ðFfin K Fin Þ=ðFmax K Fin Þ

(1)

where Fin and Ffin represent the initial and the final (steadystate) values of the fluorescence intensity before and after toxin addition, respectively. Experiments were carried out in the presence of Hg2C (50 mM), DNDS (100 mM) or Ca2C(500 mM). 2.12. Intrinsic St II fluorescence in presence of additives Fluorescence intensity and wavelengths of maximal intensity (excitation at 295 nm) was measured in the absence and the presence of several additives. Measurements were carried out in a Shimadzu spectrofluorimeter (Japan) employing a 1 cm path length cell. Additives (Hg2C, Ca2C, Ba2C and PEG 6000) were tested at the concentrations employed in the hemolytic assays. Furosemide, nitrendipine, DNDS and DIDS were not employed in this assay since their absorbances precluded a meaningful evaluation of their effect upon the toxin intrinsic fluorescence.

3. Experimental results The main red blood cells channels and transporters potentially active in St II mediated lysis and KC efflux are briefly described in Table 1. We have tried to inhibit most of these transport mechanisms in order to assess their relevance in the KC efflux and lysis that follow St II addition. 3.1. Role of Aquaporins Aquaporins are passive water channels present, at least as AQP1 and AQP3, in human RBC. In these cells, AQP1 is the most important water channel, and its function can be inhibited by Hg2C (Agre, 2000). We have performed measurements of the hemolytic activity (HA) and rate of KC efflux promoted by St II in presence of 50 mM Hg2C. The presence of this ion elicited a significant increase, both in the rate of hemolysis and KC efflux (Fig. 1 and Table 2). Similar results were obtained in five different RBC samples. This behavior takes place irrespective of the toxin concentration (data not shown). Under these conditions, the effect of Hg2C is most likely specific, since considerably higher concentrations of other Me2C ions (such as Zn2C)

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Table 1 Main channels and transporters in RBC potentially active following St II addition Channel Aquaporin 1 Band-3 Gardos (Ca2C activated KC channel) NaC/KC/2ClK co-transporter NaC/KC pump KC/ClK channel NaC/KC non-selective, voltage activated KC(NaC)/HC exchanger

Characteristics

Inhibition 2C

Ref.

Water channel 20,000 channels/cell Selective for anions 106/cell 14–40 pS, 120/cell

Hg DIDS, DNDS Nitrendipine

Usually stimulated by cell shrinkage and in a few cases by cell swelling ATP dependent Relevant mechanism of RVD in cells with high internal ClK Requires C30 mV hiperpolarization

Bumetanide

Agre (2000) Cabantchik and Greger (1992) Chandy et al., (2001) and Ellory et al. (1992) Haas (1989)

Ouabain Furosemide

Lang et al. (1998) Bursell and Kirk (1996)

–

Kaestner et al. (1999)

Relevant in hypotonic conditions; 2–3 pS

–

Kaestner et al. (1999)

have only negligible effects (data not shown). Furthermore, at considerably higher Hg2C concentrations the effect of this ion is reversed, a result that could be associated to an (unspecific) effect upon the membrane properties (Suwalsky et al., 2000). If it is assumed that the effects observed in Fig. 1 are due to AQP1 blockage, it should be concluded that reducing the rate of water transport increases both the rate of KC efflux and RBC disruption. However, this last conclusion is particularly surprising, since the lysis is ultimately associated to the water influx and could be expected that reducing this rate (by AQP1 blockage) would decrease the rate of the lytic process. Similarly, since AQP can transport cations, particularly KC (Saparov et al., 2001), it could also be expected a reduction of the rate of KC efflux in the presence of Hg2C, a result opposite to that shown in Fig. 1. This apparent anomaly could indicate a positive effect of Hg2C ions upon St II pore forming efficiency and/or a decrease of the membrane stability. This last effect is supported by experiments of osmotic fragility that show that the presence of Hg2C ions decrease the RBC resistance to an osmotic shock (Fig. 2). The data of this figure show that the NaCl concentration at which 50% hemolysis of the RBC population significantly increases from 0.38G0.03 to 0.63G0.11 g/dL in the presence of 50 mM HgCl2 (Wilcoxon test, pZ0.01). We attempted to evaluate the effect of Hg2C upon the pore forming ability of St II by measuring the rate of carboxyfluorescein release (Tejuca et al., 1996). In the presence of 50 mM Hg2C the rate of the fluorescence probe efflux increases in approximately 20% (Fig. 3), suggesting that this ion facilitates the rate of insertion and/or ensemble of the protein into the lipid bilayer. This result could be a consequence of a direct effect of Hg2C on St II structure and/or its destabilizing action on the lipid bilayer (Suwalsky et al., 2000), favouring pore formation. However, no significant changes in the toxin fluorescence spectra were observed in the presence of 50 mM Hg2C (data not shown). This, and the above mentioned results regarding the effect of

Fig. 1. Effect of HgCl2 (50 mM) upon the activity of St II (0.56 nM) added to a red blood cell suspension. (A) Time profile of the lysis elicited in a red blood cell suspension. (C) control experiment; (:) in the presence of HgCl2. (B) Time profile of KC efflux from a red blood cell suspension. (C) control experiment; (:) in the presence of HgCl2. Bars correspond to the standard deviation from duplicate experiments.

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Table 2 Effect of water, cations, and/or chloride transport manipulation on the KC efflux and lysis induced by St II Treatment

Expected effect

Observed effect on the rate of KC efflux

Observed effect on the hemolysis rate

Hg2C addition Replacing external NaC for KC Ba2C addition Bumetanide addition Furosemide addition Ouabaine addition Nitrendipine addition Ca2C increase (pCaZ3.2) DIDS (or DNDS) addition Replacing external chloride by nitrate PEG 6000

AQP1 blockage Reduces (revert) the KC flux Inhibition of KC transport NaC/KC/2ClK co-transporter blockage KC/ClK co-transporter blockage NaC/KC pump blockage Ca2C activated KC channels blockage Favors activation of KC transport Blockage of banda-3 Reduces anion transport

Increase Not measurable Decrease Unchanged Decreased Unchanged Decrease Increase Decrease Decrease

Increase Increase Increase Unchanged Increased Unchanged Decrease Increase Decrease Decrease

Increases osmolarity

Decrease

Total protection

Hg2C on the osmotic fragility of the RBC (Fig. 2) could imply that this cation affects the membrane stability and that its effect upon the HA of the toxin is considerably more complex than a simple blockage of aquaporins water channels. 3.2. Role of KC transport KC efflux through activated channels is usually a defense mechanism aimed to reduce the rate of water influx in conditions of osmotic imbalance (Guizouarn and Motais, 1999). The early efflux of KC that takes place during the lytic process promoted by cytolysins is then not surprising (Macek et al., 1994; Martı´nez et al., 2001). This passive efflux can occur through the formed pore and/or through one (or several) of the numerous mechanisms that can be activated by the swelling associated to the pore formation. In order to evaluate the role of these last processes we have

Fig. 2. Effect of HgCl2 (50 mM) addition on the osmotic fragility of a red blood cell suspension. (C) control experiment; (:) in the presence of HgCl2. Bars indicate the standard deviation from duplicate experiments.

carried out experiments employing several channel or transporter inhibitors. Also, we have performed experiments in which the external NaC was replaced by KC in order to decrease (and actually revert) the magnitude of the KC electrochemical potential gradient. The replacement of NaC by KC in the external medium induced a significant increase of St II HA, reducing t50 parameter in more than 60%, particularly when NaC and KC concentration were equivalent in the medium (Fig. 4 and Table 3). This result indicates that KC efflux is important to control the increase volume as a result of St II pore formation. Ba2C ions have been used as blockers of KC transport in erythrocytes (Hoffmann et al., 1993; Jiang and MacKinnon, 2000). The presence of this ion (up to 400 mM) does not

Fig. 3. Effect of HgCl2 (50 mM) on carboxyfluorescein release from ePC:SP LUVs following St II (20 nM) addition. Lipid concentration: 2 mM. (C) control experiment; (:) in the presence of HgCl2. f is the fraction of vesicles that have lost their encapsulated carboxyfluorescein at the indicated time.

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Fig. 4. Influence of KC gradient upon the hemolysis of a red blood cells suspension elicited by St II (0.56 nM) addition. Salt in the external buffer: (C) Control, 145 mM NaCl; (6) 70 mM NaCl and 70 mM KCl; (,) 140 mM KCl. Bars indicate standard deviations of duplicate (nZ4) experiments.

modify the toxin fluorescence (data not shown), but reduces the rate of KC efflux and increases the RBC lysis elicited by St II addition (Fig. 5 and Table 2). These results support the proposal that decreasing the KC efflux increases the rate of the lytic process. This reduced KC efflux is compatible with the blockage of one or more of the endogenous KC channels. The lack of effect elicited by bumetanide or ouabain addition would indicate that KC transport by the NaC/KC pump or the NaC/KC/2ClK co-transport are not relevant, both for the KC efflux following St II addition and the consequent cell lysis. On the other hand, furosemide addition decreases the rate of KC efflux and increases the rate of hemolysis (Fig. 6 and Table 2), a result compatible with a role of the KC/ClK co-transporter as a defense mechanism against the RBC swelling promoted by St II addition. We do not attempt to manipulate the NaC/KC non-selective voltage activated channel and the KC(NaC)/proton exchanger since they operate under hyperpolarization or hypotonic conditions, respectively (Kaestner et al., 1999).

Fig. 5. Effect of Ba2C upon the rate of KC efflux and hemolysis elicited by St II (0.56 nm) addition to a RBC suspension. Data are given as the ratio between the t50 measured in the presence of the additive and in control experiments.

Since Gardos channels have been shown to be an important defense mechanism (Lang et al., 2003) we attempted to evaluate their role by using nitrendipine and by manipulating the levels of free Ca2C ions. Interpretation of results obtained in the presence of nitrendipine are not straighforward. Addition of 10 mM nitrendipine decreases the rate of KC efflux, a result compatible with the blocking

Table 3 Effect of KC gradient upon the HA induced by St II Electrolite external composition

HA (t50, min)

145 mM NaCl 70 mM NaCl C70 mM KCl 140 mM KCl

17.2G0.6 5.7G0.5 (*) 4.5G0.4 (*)

Protein concentration employed: 0.56 nM. t50: is the time required to reduce the RBC suspension turbidity in 50%. Results represent the meansGSD from four determinations. (*) Significantly different from control value (p!0.001).

Fig. 6. Effect of additives upon the rate of KC efflux and hemolysis elicited by St II (0.56 nm) addition to a RBC suspension. Data are given as the ratio between the t30 measured in presence of the additive and in control experiments. Bars indicate standard deviations of four independent experiments. t30 corresponds to the time required to the efflux of 30% of the total cytosolic KC ions or to disrupt 30% of the cell ensamble. Additive concentrations: Furosemide: 2 mM; pCa: 3.2 (0.6 mM); DIDS: 50 m M; nitrate: 45 mM nitrate plus 95 mM chloride instead of 140 mM chloride; nitrendipine: 10 mM.

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of the KC transporting channel. However, a significant decrease is also observed in the lysis rate (Fig. 6 and Table 2). This result would suggest that nitrendipine is also interfering with St II insertion and/or pore forming capacity. Furthermore, it is interesting to point out that an activation of the Gardos channels by StII, as proposed to occur following the interaction of vibrio parahaemotlyticus haemolysin with human erytrocytes (Lang et al., 2004) cannot explain the decrease in hemolytic efficiency associated to nitrendipine addition. Increasing the free Ca2C external concentration (pCa 3.2) notably increases St II lytic efficiency and KC efflux rate (Fig. 6 and Table 2). These results would suggest that the role of Ca2C is more complex than activation of the Gardos channel and could be explained in terms of a promoting action of Ca2C upon St II lytic action. The enhanced HA and KC efflux could result from a direct Ca2C-toxin interaction and/or be due to the destabilizing effect of this ion influx associated to disturbance of the RBC membrane (Bevers et al., 1999). This could favor a better accommodation of the protein into the membrane, since lipid rearrangement has been reported to be involved in actinoporin–membrane interaction mechanism (AlvarezValcarcel et al., 2001; Bonev et al., 2003; Anderluh et al., 2003). Interestingly, attempts to test direct activation of the toxin by Ca2C in model membranes were unsuccessful since addition of 500 mM Ca2C to PC:SM liposomes reduces in 30% the rate of intravesicle trapped carboxyfluorescein efflux. Furthermore, the presence of mM concentrations of Ca does not modify the toxin fluorescence spectra (data not shown). This would suggest that enhancement of the toxin activity by Ca2C is more due to membrane destabilization by its entrance through St II pores and activation of molecular processes present in cells (and not in vesicles) than to an increased toxin activation resulting from their direct interaction. 3.3. Role of chloride transport Chloride transport can be important in itself (increasing the cytosolic osmolality and/or decreasing intracellular pH) and/or by its effect in NaC and/or KC co-transport. In order to evaluate the influence of chloride transport in KC efflux and the lytic process induced by St II, ClK was replaced by the less transportable nitrate anion. Also, the rate of ClK transport was reduced by blockage of the band-3 system, employing DIDS or DNDS as inhibitors (Cabantchik and Greger, 1992). Results obtained in the presence of DIDS (50 mM) and summarized in Fig. 6 and Table 2, show a decrease of KC efflux and HA induced by St II addition to the cell suspension. Similarly, the presence of DNDS (100 mM) reduced to one half the rate of the lytic process (data not shown). Replacement of chloride by nitrate also provoked a decrease, both in KC efflux and HA (Fig. 6 and Table 2). It can be concluded then that decreasing the anion transport (either by band-3 blockage or by replacing ClK by

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the less transportable nitrate anion) produces a noticeable decrease in both the KC efflux and the rate of the lytic process. In particular, the effect upon the KC efflux indicates that anion transport is relevant even at early stages of the lytic process. The above mentioned results are compatible with a relevant role of band-3 in the anion transport required to allow the KC efflux and the entrance of electrolyte that generates the osmotic imbalance leading to the RBC disruption. However, an alternative explanation could be that the employed anion exchange blockers can modify the efficiency of pore formation by the toxin. Similarly, if anion transport takes place through the toxin pore and is the ratelimiting step, it could be expected that the less mobile nitrate ion could be transported slower than chloride ions. In order to test if anion transport inhibitors interfere with St II pore forming capacity, we performed liposome permeabilization experiments in presence of 100 mM DNDS. The results obtained show a significant decrease (by a factor two) in the rate of carboxyfluorescein efflux following the toxin addition. This reduction would indicate that St II pores formation and/or function could be unpaired by DNDS. 3.4. Addition of a non-permeating solute In order to further assess the osmotic character of the lytic process induced by St II we have evaluated both KC efflux and lysis rate in presence of PEG 6000. Typical results are shown in Fig. 7. These data show that PEG addition affects more the lytic process (which is almost totally prevented, Fig. 7A) than the rate of KC efflux (Fig. 7B). These results show that pore formation takes place even in hyperosmotic conditions, and that the lack of lysis observed under these conditions results from a reduced osmotic gradient that minimizes the entrance of small ions (NaC, ClK) to the RBC cytosol. The reduced rate of KC efflux is consistent with the slower rate of pore formation observed in hyperosmotic conditions by following the exit of carboxyfluorescein from PC-SM unilamellar liposomes (data not shown). Furthermore, a small decrease in the intrinsic fluorescence of the protein (17%) elicited by PEG (10 mM) addition could indicate some degree of protein conformational change in the presence of the additive.

4. Discussion St II exerts its hemolytic action by forming hydrophilic pores in the cell membrane with an average radius of 1.1 nm and, consequently, cell lysis results from a colloidal osmotic shock (Tejuca et al., 2001). Along an outward osmotic gradient of non-permeant molecules, such as hemoglobin, net influx of water increases cell volume until cell membrane disruption (MacGregor and Tobias, 1972). The occurrence of a significant KC efflux observed with non-permeating solutes under conditions of minimum lysis

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The lytic process mediated by water influx as a consequence of an osmotic imbalance must necessarily involve three successive and/or simultaneous distinct processes: cationic transport (KC efflux and NaC influx under the present experimental conditions), anion transport (mainly ClK intake under the experimental conditions) and water influx. In order to assess the main transport pathways active during the lytic process induced by St II, we tried to manipulate water, cationic and chloride main transporters (Tables 1 and 2). 4.1. Hg2C effects and the blockage of AQP1 water transport It has been reported that Hg2C(50 mM) blocks AQP1 in RBC (Tsai et al., 1991), a channel efficient in water and cations transport, although the relevance of this last process is open to discussion (King et al., 1996; Saparov et al., 2001). If AQP1 transport is blocked, it could be expected a decreased rate of lysis if the limiting process is the rate of water entrance and it mainly takes place through AQP1. However, the present data (Fig. 1 and Table 2) show that Hg2C addition increases the rate of KC efflux and RBC lysis. In particular, the increased rate of hemolysis is not compatible with the expected response to blockage of the water influx, indicating than the effect of Hg2C must be more complex than a simple reduction in water flux due to its interaction with AQP1. This conclusion is supported by an increased RBC osmotic fragility in the presence of Hg2C (Fig. 2) and a potentiation of carboxyfluorescein release in liposomes induced by St II when this ion is present in the medium (Fig. 3). The present results would indicate that, at least in presence of Hg2C ions, water influx would still efficiently take place in spite of AQP1 blockage. Fig. 7. Effect of PEG (10 mM) in the external medium upon lysis and KC efflux from a red blood cells suspension exposed to St II (0.9 nM). (A) Effect of PEG on the hemolytic activity. (C) Control; (:) in the presence of PEG. The ordinate represents the percentage of the initial turbidity (measured at 600 nm) that remains after the indicated time. (B) Effect of PEG on KC efflux. (C) Control; (:) in the presence of PEG. Values correspond to the average of three measurements.

(Fig. 7), is compatible with this interpretation of the lytic process provoked by St II. The aggression of the toxin to the cell permeability barrier allows the transmembrane passage of ions, mainly NaC, KC and ClK (Tejuca et al., 1996). In agreement with this, previous studies have shown that insertion in membranes of St II (Martı´nez et al., 2001) or the actinoporin Equinatoxin II (Eq TII) (Macek et al., 1994) provoke an efflux of KC previous to lysis. This ionic flux can take place through the newly formed St II-pores and/or the endogenous mechanisms acting in the regulatory volume decrease (RVD) that takes place after cell swelling.

4.2. KC efflux as a defense mechanism The results obtained in the presence of Ba2C (Fig. 5) and when external NaC is changed by KC (Fig. 4 and Table 3) show that KC efflux can be considered as a defense mechanism associated to the volume increase resulting from the toxin pore formation. The mechanisms by which cell swelling activates volume-sensitive channels are poorly understood. One of the predominant pathways for the RVD in mammalian cells faced to swelling is the opening of KC and anion channels. The KC channels involved are usually large-conductance, Ca2C-activated channels, but other channels have also been claimed to play a role (O’Neill, 1999). For example, the NaC/KC/2ClK co-transporter has been considered to play an important role in cell ion homeostasis and volume control (Flatman, 2002). Nevertheless, our results indicate that KC efflux does not proceeds through the NaC/KC/2ClK co-transporter since addition of bumetanide, a specific inhibitor of this transport (Gulbins et al., 1997) did not change neither KC efflux nor hemolysis rate. Even though this transporter can move ions in both directions, this result is not surprising since the NaC/KC/

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2ClK co-transporter operation has been mainly associated to the regulatory volume increase taking place after cell shrinkage (Flatman, 2002; O’Neill, 1999). An additional mechanism that mediates RVD is activation of the KC–ClK co-transporter which transports KC and ClK stoichiometrically in either direction across the plasma membrane. Although structurally related to the NaC/KC/2ClK co-transporter and inhibited by furosemide, this transporter is NaC independent and insensitive to bumetanide, it has a strict requirement for ClK, and is the principal mechanism for RVD in cells with high ClK intracellular concentrations, such as erythrocytes (O’Neill, 1999). The present results, summarized in Figs. 5 and 6, as well as in Table 2, would indicate that this channel could play a role in KC efflux and represents a protection mechanism of the red blood to retard its St II induced disruption. Ca2C ions constitute a signal for the activity of the so called Ca2C-activated KC channels (O’Neill, 1999). Volume regulation via Ca2C-activated KC channels in isosmotic swelling has been reported (Brayden et al., 1992). In the present work a dominant role of Ca2Cactivated KC-channels on KC efflux induced by St II is not supported by data obtained in presence of nitrendipine (Fig. 6 and Table 2). This inhibitor provoked a slight decrease of KC efflux but also a decrease of the hemolysis rate in relation to the non-treated cells. These last results are contrary to that expected since hampering the compensatory KC efflux would imply a faster volume increase and consequently a high lytic rate. Furthermore, as the increase of KC efflux and hemolytic activity induced by Ca2C addition (Fig. 6 and Table 2) could not be ascribed to the direct effect of this ion upon the protein, it is proposed that the entrance of this ion to RBC cytosol through the pore formed by St II, leads to the cell destabilization (Bevers et al., 1999) favoring the lytic process. Taking together, the results summarized in Table 2 would indicate that, under the present experimental conditions, KC/ClK co-transport could play a role in the RVD by decreasing the intracellular K C and ClK concentrations. This could afford a defense mechanism against the osmotic imbalance produced by the pore formation. 4.3. Effect of DIDS or DNDS addition and reduced chloride gradient The data summarized in Fig. 5 and Table 2 would indicate that anion transport is a limiting factor, both in the rate of KC efflux and in the rate of disruption of the RBC ensemble. In fact, the presence of DIDS (or DNDS), blockers of band-3, and the change of chloride by the less mobile nitrate, reduce both the rate of KC efflux and the rate of the lytic process. The results of KC efflux would indicate that this process is sided by chloride transport, and that

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diminishing the rate of one of them reduces the rate of the other. With regard to the retarding effect on the lysis, the present data show that massive NaC influx, necessary to generate the osmotic imbalance, must take place parallel to anion influx, in order to avoid depolarization of the cell (Staines et al., 2001). All the data obtained regarding anion transport manipulation is compatible with a role of band-3 in this process. In addition, intake of ClK through the cotransporter band-3 could take place by efflux of the intracellular bicarbonate, that would decrease the intracellular pH with a concomitant decrease in the membrane stability (Sato et al., 1993). This last effect could contribute to the observed hemolysis promoted by St II addition. An alternative interpretation is that anion transport takes place exclusively through toxin channels. These channels, although cation selective, retain some capacity for anion transport (Tejuca et al., 1996). A predominant role of St II pores in the anion transport should explain the present data only if they were also blocked by DNDS and DIDS. These inhibitors bind to lysine groups and are rather general blockers of anion transporters (Cabantchik and Greger, 1992). The reduced rates observed in KC transport and lysis in the presence of nitrate could then simply result from the smaller mobility of this larger ion through St II pore. However, the effect of changing chloride by nitrate seems to be larger (a factor 1.5 when 32% of the chloride is replaced by nitrate) than that expected from their relative mobility, the limiting factor for permeation through a large pore such as that formed by St II. In fact, the mobility of chloride ions is only 1.05 times that of nitrate. Taking together the present results would suggest that, at least partially, anion transport is band-3 mediated and that anion transport blockers, such as DNDS, besides blocking this anion exchanger can reduced the rate of formation and/or the efficiency of toxin derived channels. Regarding the possible role of endogenous channels in RBC in the cytolytic process induced by St II we can conclude that AQP1 activity is not essential for the occurrence of the lytic process provoked by St II addition. Furthermore, KC efflux as a defense mechanism associated to the volume increase induced by St II, is not related with the activation of Ca2C dependent KC channels. However, KC/ClK co-transport participates at least partially in the KC efflux induced by St II and chloride influx, a process that can regulate both KC efflux and lysis induced by the toxin, is partially mediated by the endogenous cell transporters. In particular, the results are compatible with a role of band-3 in this process.

Acknowledgements This work is part of a Chile–Cuba collaboration program and has been financed by FONDECYT (Project 1030033) and the UNU-BIOLAC fellowship program.

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