©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Chloride Conductive Pathways Which Support Electrogenic H Pumping by Leishmania major Promastigotes (*)

(Received for publication, November 7, 1994; and in revised form, December 16, 1994 )

Lita Vieira (1)(§) Itzchak Slotki (2) Z. Ioav Cabantchik (1)(¶)

From the  (1)Department of Biological Chemistry, Institute of Life Sciences, Hebrew University, Jerusalem 91904 and the (2)Renal Unit, Shaare Zedek Medical Center, P. O. B. 3235, Jerusalem 91931, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The proton extrusion mechanisms of Leishmania promastigotes were studied in terms of electrogenic movements of protons and anions (Cl and HCO(3)). Changes in membrane potential (V) and intracellular pH (pH) were monitored fluorimetrically with the potential sensitive dye bis-oxonol and the pH-sensitive dye tetraacethoxymethyl 2`,7`-bis-(carboxyethyl)-5,6-carboxyfluorescein, respectively. In nominal bicarbonate-free medium (pH7.4, 28 °C), V and pH of Leishmania promastigotes were maintained at -113 ± 4 mV and 6.75 ± 0.02, respectively. In Cl free (gluconate-based) medium, cells underwent a time-dependent acidification (0.3 pH units) and a long term membrane hyperpolarization (7-10 mV), both of which were greatly enhanced in the presence of the anion blocker, 4,4`-diisothiocyanodihydrostilbene-2,2`-disulfonic acid (H(2)DIDS). Cells in Cl-free medium underwent a marked depolarization upon treatment with the H-ATPase inhibitor dicyclohexylcarbodiimide (DCCD), but hyperpolarized after repletion with Cl. In Cl-depleted cells, replenishment of Cl led to a H(2)DIDS-sensitive cytoplasmic alkalinization and a small initial hyperpolarization. Cells exposed either to DCCD or to the H uncoupler carbonylcyanide chlorophenylhydrazone caused a marked cytoplasmic acidification and membrane depolarization. In the presence of 25 mM HCO(3), promastigotes maintained an almost neutral cytosol, irrespective of H pump action or ionic composition of the medium. The present observations provide evidence for the operation of a DCCD-sensitive electrogenic H-ATPase which contributes to the maintenance of a highly hyperpolarized plasma membrane in Leishmania promastigotes. H pump activity required a parallel pathway of Cl ions in order to dissipate the pump generated electrical potential. In nominally CO(2)-free media, the two electrogenic systems are implicated in the maintenance of cell pH and indirectly in electrochemically driven nutrient uptake. In physiological CO(2)/HCO(3)-containing media, the H pump and Cl channel play a role only secondary to that of HCO(3) in pH homeostasis.


INTRODUCTION

Leishmania parasites alternate between two major developmental stages, the flagellated promastigote which lives in the midgut of the sandfly vector and the obligatory intracellular nonmotile aflagellated amastigote which lives in the phagolysosome of mammalian macrophages. The successful adaptation and survival of the parasite in these distinctly different and extreme environments depends on the ability of these cells to acquire nutrients from the neighboring environment and in the maintenance of a constant internal milieu(1) .

In plants and lower eukaryotes, nutrients are acquired by membrane transport mechanisms which involve coupling of substrate with uptake of H down their electrochemical gradient(2, 3) . The role of an electrogenic H-translocating ATPase in nutrient uptake has been more conclusively demonstrated in bacteria(4) , fungi(5) , and plants(2, 6) . An analogous H-ATPase has been proposed in promastigotes of Leishmania(7, 8) , but direct experimental evidence for H pumping at the cell level has been lacking. A role for a putative H-pumping mechanism in intracellular pH regulation has been previously hypothesized on the basis of experiments carried out in nominally CO(2)-free media(7, 8) , but the notion of an indirect contribution of such a mechanism in H-coupled nutrient uptake has remained controversial(9, 10) . Moreover, since Leishmania promastigotes naturally propagate in a HCO(3)-rich environment, it is not clear to what extent H pumping subserves the maintenance of cell pH.

Presently, little is known about the relative permeabilities of the parasite plasma membrane to the prevailing ions Na, K, H, Ca, HCO(3), and Cl, let alone about passive or active ionic membrane conductances. In this work we explored the electrogenic components underlying H-pumping mechanisms of intact Leishmania major promastigotes, that is H pumping and anionic conductances. The studies were carried out with intact cells placed in nominally CO(2)-free media of different ionic composition and with the aid of the potentiometric fluorescent dye, bis-oxonol(11) . Inhibitors of higher eukaryotic H pump and Cl channels (12, 13) were used as tools for assessing the respective contributions of both cationic and anionic transport systems to Vand pH. The studies indicate that both, a DCCD(^1)-sensitive H pump and a 4,4`-diisothiocyanodihydrostilbene-2,2`-disulfonic acid (H(2)DIDS)-sensitive Cl channel contribute electrogenically to acid secretion and to the maintenance of a hyperpolarized plasma membrane and suggest a role for the two systems in pH homeostasis and H-coupled nutrient uptake in nominally CO(2)-free media. In the more physiological HCO(3)-containing media, while the electrogenic role of the pump is maintained, pH regulation is primarily supported by transporters of HCO(3).


EXPERIMENTAL PROCEDURES

Materials

Bis-(1,3-diethylthiobarbituric acid) trimethine oxonol (DiSBAC2(3)) (bis-oxonol), tetraacethoxymethyl 2`,7`-bis-(carboxyethyl)5,6-carboxyfluorescein (BCECF), gramicidin, and H(2)DIDS were procured from Molecular Probes. RPMI media, carbonylcyanide chlorophenylhydrazone (CCCP), and DCCD were from Sigma (Israel), and fetal calf serum was purchased from BioTechnologies Inc. (Beth Haemek, Israel). All other chemicals were of highest available grade of purity. They were obtained from Sigma (Israel).

Solutions

The composition of Cl medium was 137 mM NaCl, 4 mM KCl, 1.5 mM KH(2)PO(4), 8.5 mM Na(2)HPO(4), pH 7.4, supplemented with 20 mM HEPES, 11 mM glucose, 1 mM CaCl(2), and 0.8 mM MgSO(4). Cl-free medium was similarly prepared but with gluconate substituting for Cl. N-Methylglucamine-Cl (NMGCL) medium contained 140 mM NMGCl, 11.1 mM glucose, 0.8 mM Mg(2)Cl, 1 mM CaCl(2), and 10 mM HEPES, pH 7,4. Stock solution of 1 M potassium gluconate (KGlu) was supplemented with 10 mM HEPES, pH 7,4. Ca-free medium had the same composition as the Cl medium except that 0.5 mM Na-EDTA replaced the 1 mM CaCl(2). HCO(3) medium was essentially the same as Cl or gluconate medium, except that NaHCO(3) (25 mM) replaced the appropriate fraction of the respective sodium salt of Cl or gluconate. Stock solution of 1 M sodium gluconate (NaGlu) was prepared as KGlu but K was substituted by Na. The osmolarity was checked using a Wescor osmometer (Wescor Inc., Logan, UT). All solutions were isotonic (290-300 mosm) and nominally CO(2)-free.

Parasites

Promastigotes of L. major strain LCR-137 were grown in RPMI medium (Sigma) supplemented with 10% fetal calf serum, 25 mM HEPES, and 25 mM sodium-bicarbonate at 29-30 °C. The cells were harvested in exponential growth phase (2-3 times 10^7 cells ml) by centrifugation at 2,500 times g for 10 min at 4 °C and were washed twice with isotonic phosphate-buffered saline, pH 7.4, supplemented with 11 mM glucose, 0.8 mM MgSO(4), 1 mM CaCl(2). Cells suspension were kept at room temperature until further use.

Measurements of Cytoplasmic pH (pH(i)) and Membrane Potential (V(m))

pH(i) determinations were based on ratio fluorescence measurements with BCECF, as described previously (14) . V(m) was assessed fluorimetrically as described by Rink et al.(11) . Bis-oxonol was added from a 100 µM stock solution in dimethyl sulfoxide (Me(2)SO) to the indicated medium to a 1:1000 dilution. Aliquots of 5 times 10^6 cells ml were transferred to a plastic cuvette containing 2 ml of buffer containing the dye, and sitting in a thermostatically controlled (28.1 ± 0.1 °C) and magnetically stirred cuvette holder. Calibration of V(m) was done in cells suspended in isotonic NMGCl, pH 7.4, and in the presence of the Na/K ionophore gramicidin D (0.8 µM). After signal stabilization was achieved, increasing KCl concentrations were added to the medium and the fluorescence signal was recorded. In these conditions the transmembrane potential (V(m)) is given by V(m) = -59.4 log([K](i)/[K](o)), where i and o indicate the internal and external concentrations of K, respectively, and [K](i) was taken as 120 mM(15) . It was assumed that [K](i) remained constant during brief cell treatments. The relationship between the fluorescence signal and the calculated V(m) for the different [K](o) was nearly linear in the range of 0 to -80 V(m), and although non-linear beyond that range, it could still be used for calibration as indicated by Rink et al.(11) (Fig. 1). The fluorescence signal of bis-oxonol per se was affected neither by the anion blocker, H(2)DIDS (in the absence of cells) nor by the ionic contents of the medium. However, since some gramicidin preparation raised the background fluorescence of bis-oxonol-containing solutions, background values were subtracted from the original recordings. Fluorescence measurements were carried out in a PTI fluorescence station (PTI, GmbH, Wedel, Germany). For bis-oxonol we used an excitation wavelength of 540 nm (slit width 4 nm) and an emission wavelength of 580 nm (slit width 10 nm). For measurements done in the presence of HCO(3), the cuvettes were filled with liquid and tightly sealed.


Figure 1: Calibration of membrane potential V in Leishmania promastigotes. An aliquot of 5 times 10^6 cells/ml was equilibrated with 0.1 µM bis-oxonol in NMG solution. The traces are of fluorescence intensity (540 excitation and 580 nm emission) after background substraction following addition of gramicidin (0.8 µg/ml) and KCl to give the final indicated concentrations (inset). The main graph depicts the relationship between fluorescence change (F) and the calculated K equilibrium potential V, in millivolts (mV).



The ATPase inhibitor DCCD (10 µM), the protonophore CCCP (1 µM), and the anion transport inhibitor H(2)DIDS (0.5 mM) were added directly to the medium containing cells and fluorescent dye prior to measurements. None of these inhibitors or uncoupler interfered with the fluorescence signal of bis-oxonol.

ATP Measurements

ATP was measured using the luciferin/luciferase method(16) . Briefly, 0.5 times 10^8 cells/ml were freeze-thawed nine times in PBS buffer, pH 7.4, supplemented with 11 mM glucose, 1 mM CaCl(2), 0.8 mM MgSO(4). A 50-µl aliquot of cell suspension was treated with 5% trichloroacetic acid for 15 min on ice, centrifuged, and the supernatant diluted 1:40 in 25 mM HEPES buffer, pH 7.4. A 200-µl aliquot was added to a cuvette and rapidly mixed with 40 µl of luciferin-luciferase reagent (Lumac/3 M). Chemiluminescence was read in a Lumac/3M (Biocounter. Inc.). Data shown are of representative experiments which were conducted independently under the same conditions at least five times, or as otherwise indicated.


RESULTS

Resting Membrane Potential (V(m))

V(m) of Leishmania promastigotes was studied in various media with the aim of assessing the contribution of the various physiological ions to the electrical properties of the plasma membrane. The measurements of V(m) were based on the use of the anionic lipophilic bis-oxonol, which partitions between membrane and adjacent aqueous regions in a potential dependent manner(17) . The experimental system allowed on line fluorescence measurements of V(m) which reflected changes in the electrical properties of the membrane. Other cationic fluorescence dyes were found inadequate for use with Leishmania parasites. In normotonic conditions at 28.1 ± 0.1 °C, pH(e) 7.4, the steady state V(m) estimated from the calibration curve (Fig. 1) was -113 ± 4 S.E. A similar V(m) value was previously reported for Leishmania donovani promastigotes, based on partitioning of the radiolabeled lipophilic cation tetraphenylphosphonium (TTP) between cells and medium(18) .

Role of Cations

The contribution of ionic potassium, sodium, and calcium conductances to the highly negative V(m) of cells was initially assessed in terms of effects of individual ions on V(m). K-conductance (g(K)) was expected to make a major contribution to the V(m) of -113 mV, based on the calculated K-equilibrium potential (E(K)) of -80 mV and previous potentiometric studies with TPP(18) . However, as seen in Fig. 2, the fluorescence intensity of bis-oxonol was hardly affected either by addition of KGlu or NaGlu to the medium (Fig. 2). Removal of Ca from the medium induced only a minor depolarization (<10 mV). However, major fluorescence changes occurred only if the channel former gramicidin (0.8 µM) (Fig. 1) or the calcium ionophores ionomycin or A23187 (not shown) were added to the cells. This suggests that, in the given experimental conditions, neither K, Na, nor Ca conductances contributed substantially to V(m). These results differ from those obtained with L. donovani in which K was implied to play some role in setting V(m)(18) . Methodological differences may account for the discrepancy (see ``Discussion'').


Figure 2: Effect of cations on V. Cells were suspended in NMGCl medium, pH 7.4, previously equilibrated with 0.1 µM bis-oxonol. After signal stabilization was achieved, increasing concentrations of either KGlu (potassium gluconate) or NGlu (sodium gluconate) were added. V was measured as described in the legend of Fig. 1. Traces are representative of three separate experiments.



Role of Anions

The role of Cl and HCO(3) ions was initially assessed by following both V(m) and pH(i) in cells preincubated in Cl-free media. In a previous study we indicated that, in such conditions, cells undergo a time-dependent acidification as a result of intracellular depletion of Cl which in turn limits pump-mediated acid removal(14) . In Fig. 3we show that during the acidification phase, the membrane undergoes a time-dependent hyperpolarization, presumably reflecting the diffusion potential generated by the pump. The V(m) values attained in the presence of the inhibitor were only approximate, since the calibration curve was non-linear in the range below -80 mV (the values were obtained by interpolation from the calibration curve shown in Fig. 1). Upon readdition of Cl to the medium of Cl-depleted cells, the membrane swiftly hyperpolarized, and the cytosol underwent a marked alkalinization (Fig. 4). The Cl-induced hyperpolarization was relatively small (from -122 mV to 128 mV), reached a plateau at about 7 min and was followed by a slow depolarization (not shown) to the physiological V(m). The hyperpolarizing effect of Cl can be interpreted to result either from an inward Cl conductance or from an initial reactivation of the H pump.


Figure 3: Effect of Cl depletion on V(m) and pH. In parallel experiments, cells were either preloaded with the pH indicator BCECF and subsequently suspended in gluconate medium, pH 7.4 or were suspended directly in gluconate medium, pH 7.4, containing bis-oxonol (0.1 µg/ml). pH and V were continuously monitored as indicated under ``Experimental Procedures.''




Figure 4: Effect of Cl replenishment on the pH and V of Cl-depleted cells. Cells were preincubated in gluconate medium, pH 7.4, for 30 min at 30 °C and a fraction loaded with BCECF. Subsequently cells were resuspended in Cl medium, pH 7.4, and pH and V were recorded as indicated in Fig. 3.



Effect of Transport Inhibitors

In this study we used the anion transport blocker H(2)DIDS and the ATPase chemical modifier DCCD as well as the protonophore and energy uncoupler CCCP. Since some of these agents might interfere with the transport processes by metabolic poisoning of cells, we assessed first the effects of inhibitors on cell ATP levels. The ATP levels remained essentially constant in Cl or gluconate based media (1.52 ± 0.15 mM and 1.42 ± 0.09 mM, respectively), dropped insignificantly in the presence of H(2)DIDS (1.37 ± 0.08 mM) and decreased in the presence of either DCCD (0.70 ± 0.12 mM) or CCCP (1.0 ± 0.17 mM). Cells suspended in Cl medium underwent acidification and hyperpolarization in the presence of the anion transport blocker H(2)DIDS (Fig. 5). The marked acidification could have resulted from blockage of either the putative H pump or a putative anion channel. However, the pronounced hyperpolarization caused by H(2)DIDS in Cl medium is consistent with the notion that the H pump remains active and that the major effect of the inhibitor is on an anion conductive pathway. In order to further distinguish between blockage of H pump and inhibition of an anion channel as the cause of cytoplasmic acidification, cells were suspended in Cl medium with the ATPase inhibitor DCCD (10 µM). As shown in Fig. 6A, the chemical modification induced cytoplasmic acidification and membrane depolarization. These results are consistent with the hypothesis that a DCCD-sensitive and electrogenic pump is involved in proton extrusion from the cells, as we and others have hypothesized(7, 8, 14) . However, an indirect effect via ATP depletion could not be completely excluded. Further support for the involvement of an electrogenic H pump in the maintenance of pH(i) and V(m) in L. major promastigotes was obtained using the protonophore CCCP. The proton uncoupler, which equilibrates [H] across biological membranes(13) , induced both a robust membrane depolarization from -113 to -40 mV and a cell acidification from pH(i) 6.75 to 6.4 (Fig. 6B). The V(m) attained in the presence of CCCP was in the range of the predicted H equilibrium (Nernst) potential, consistent with the notion that the cytoplasmic acidification resulted from inward and electrogenic (CCCP-mediated) movement of Hs.


Figure 5: Effect of H(2)DIDS on pH and V. pH and V were followed fluorimetrically in cells suspended in Cl medium, pH 7.4, in the absence or presence of 0.5 mM H(2)DIDS.




Figure 6: Effect of the H-ATPase inhibitor DCCD and the protonophore CCCP on pH and V. Cells pretreated as described in Fig. 4were suspended in Cl medium, pH 7.4, either in the presence or absence of bis-oxonol. DCCD (10 µM) (A) or CCCP (1 µM) (B) were added at the times indicated by the arrows, respectively. Fluorescent changes were recorded as described in Fig. 3.



Direct evidence for the existence of a Cl conductive pathway was observed in cells in which the electrical contribution of the pump was reduced by treatment with DCCD alone (Fig. 7A) or with both DCCD and CCCP (Fig. 7B). In Cl-free medium, the marked depolarization elicited either by a single agent or both agents together, was followed by a significant hyperpolarization upon addition of NMGCl (Fig. 7). Addition of an equivalent amount of sodium gluconate produced only a minor change, due to a dilution effect. We interpret the hyperpolarization to result from an inward negative diffusion potential created by Cl supplementation, due to the existence of a Cl conductive pathway in Leishmania promastigote plasma membranes. Essentially similar results were obtained with cells suspended in isotonic mannitol-based media (not shown).


Figure 7: Hyperpolarization by addition of Cl to pump-inhibited cells. Cells suspended in gluconate medium, pH 7.4, containing bis-oxonol were treated with DCCD (10 µM), either alone (A) or with CCCP (1 µM) (B). At the indicated time NMGCl (filled squares) or NaGlu (open squares) were added (50 mM final). V was determined fluorimetrically as described in Fig. 3.



Role of HCOin pH Regulation

Since Leishmania promastigotes naturally propagate in a neutral to alkaline HCO(3)-rich environment(19) , we tested the effect of HCO(3) anions on pH(i) under a variety of experimental conditions (Fig. 8, upper). Cells preincubated in Cl medium, when suspended in the same medium, but containing 25 mM HCO(3), underwent a slow alkalinization from pH 6.73 ± 0.02 to 6.95 ± 0.05. In the presence of H(2)DIDS, alkalinization was abolished and the final steady state pH(i) attained was 6.72 ± 0.03. When cells were suspended in Cl-free (gluconate) medium, pH(i) decreased and attained a steady state value of 6.50 ± 0.03. Addition of HCO(3) abrogated the acidification and induced a slight alkalinization, but the final pH(i) attained was relatively lower (6.82 ± 0.04) than in Cl medium. A summary of steady state pH(i) values attained in HCO(3) containing and in nominally CO(2)-free media at extracellular pH 7.4 and 8.0 is given in Fig. 8(lower). The results indicate that Leishmania promastigotes can maintain a somewhat acidic cytosol (pH 6.75-6.83) in nominally Cl-free medium at either extracellular pH. Addition of up to 25 mM HCO(3) had profound alkalinizing effects, but pH(i) rose only to about neutrality. These studies clearly indicate a major, but not necessarily exclusive, role for HCO(3) in pH(i) homeostasis of promastigotes.


Figure 8: Effect of HCO(3) on pH in Leishmania promastigotes. Upper, pH was followed fluorimetrically (as shown in Fig. 1) in Cl medium (Cl), pH 7.4, or gluconate-medium (Glu(o)), pH 7.4, either in the presence or absence of 25 mM HCO(3) ± H(2)DIDS (0.5 mM). Lower, steady state pH values of cells incubated for 30 min in Cl medium at either pH 7.4 or 8.0 (pH) in the presence (+) or absence(-) of 25 mM HCO(3).




DISCUSSION

In this work we studied the electrical properties of H- and anion transport systems of Leishmania promastigotes. We based the studies on fluorescence measurements of V(m) and pH(i) with the aid of fluorescent potentiometric and pH metric dyes bis-oxonol (11) and BCECF(20) , respectively. We showed that both H and Cl extrusion from cells are electrogenic. A putative H pump is shown to be primarily responsible for generating a pronounced electronegative V(m) and a Cl channel is shown to subserve H extrusion by dissipating the high potential generated by the pump. Such coupling between H and Cl fluxes has been demonstrated in a variety of cells and in acidifying organelles of higher eukaryotes(21, 22, 23) .

The highly negative V(m) of -113 ± 4 mV found in Leishmania promastigotes in steady state conditions agrees with previous measurements of V(m) based on the lipophilic cationic dye, TPP in L. donovani promastigotes(18) . The maintenance of a relatively high negative membrane potential seems to be a common feature of several parasitic protozoa(24) , particularly in the species related trypanosoma(25, 26) . In mammalian cells, K ion gradients and K-conductance, g(K), are the major determinants of the resting cell membrane potential(27) .

In Leishmania promastigotes, a substantial contribution of g(K) to V(m) could be implied on the basis that the E(K) of -80 mV is close to V(m). If that is the case, then a 10-fold change in [K](i)/[K](o) should presumably shift V(m) by about 60 mV. The fact that V(m) changed very little (this work) or only to a minor extent (18 mV) in a previous study (18) would indicate that g(K) contribution to V(m) is small relative to other ionic conductances. Regarding g, its contribution to the resting V(m) is presumably also small. This is shown in the minor hyperpolarization which follows when cells are placed in Cl-free media. We interpret this to reflect g limiting H pumping (i.e. cell alkalinization) because of Cl depletion from cells. Thus, the role of g is in supporting cation (primarily H) extrusion. This is best exemplified in the acidification and membrane hyperpolarization which resulted from placing cells in Cl-free media or in Cl-containing medium in the presence of the anion transport blocker H(2)DIDS ( Fig. 3and Fig. 5). All this suggests that extrusion of Hs is probably the major factor behind the highly negative V(m) generated across the Leishmania plasma membrane, while the movement of anions would serve to partially dissipate this potential and facilitate further H extrusion. These notions are consistent with and supported by the effects obtained with DCCD and CCCP on both V(m) and pH(i). In this case, the partial abrogation of H pump activity by DCCD and the dissipation of H gradients by CCCP led to cell acidification and membrane depolarization. If the V(m) was partially clamped with CCCP, then addition of extracellular Cl to cells placed in gluconate medium, should also lead to some hyperpolarization. This was indeed observed (Fig. 7), indicating the presence of a Cl-conductive pathway.

Taken together, the experimental evidence provided in this work supports the parallel operation of an electrogenic H pump in conjunction with a Cl channel, whose combined action is acid extrusion. As to the physiological role of such systems, they should be evaluated in the context of the physiological media in which parasites dwell(28, 29) . Parasites are major producers of acids (30) which would tend to acidify their cytosol. The present work was carried out primarily in media not supplemented with HCO(3) (although referred to as nominally CO(2)-free, it had ambient dissolved CO(2) and HCO(3)). Blocking of acid extrusion resulted in cell acidification. However, when cells were suspended in HCO(3)-supplemented media, pH(i) rose from 6.75 to about neutrality, and it was maintained in the 6.85-7.0 range even in the presence of an anion transport inhibitor or when extracellular pH was raised to 8.0. These data suggest that the physiological role attributed to H pumping (7, 8, 14) and Cl channels (14) in regulating the cytosolic pH of Leishmania promastigotes is substantial in relatively acidic or HCO(3)-free media, but probably more modest in HCO(3)-containing media than previously hypothesized. The somewhat overestimated role of the systems found in ambient (i.e. nominally CO(2)-free) conditions is analogous to the role previously attributed to the mammalian Na-H antiporter in pH(i) changes following signal transduction in the absence of HCO(3)(31, 32) . Thus both the H pump and HCO(3) transporters are likely to play an important role in pH homeostasis of Leishmania, by mediating acid neutralization and/or extrusion. However, H pumping by promastigotes remained also fully active even in bicarbonate media, as judged by the maintenance of a highly hyperpolarized membrane.^2 This implies that the combined action of parasite H pump and Cl channel might not only be important for pH homeostasis per se. The marked membrane hyperpolarization and possible generation of a proton motive force might subserve the active uptake of substances, as previously proposed(7, 8, 19) . Moreover, as these transport systems subserve essential physiological functions, they might be considered potential targets for pharmacological intervention.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant AI-20342. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the BID-Consejo de Investigaciones Cientificas y Tecnologicas, Venezuela.

To whom correspondence and reprints requests should be addressed. Tel.: 972-2-585-420; Fax: 972-2-586-974.

(^1)
The abbreviations used are: DCCD, dicyclohexylcarbodiimide; H(2)DIDS, 4,4`-diisothiocyanodihydrostilbene-2,2`-disulfonic acid; BCECF, tetraacethoxymethyl 2`,7`-bis-(carboxyethyl)-5,6-carboxyfluorescein; bis-oxonol, bis-(1,3-diethylthiobarbituric acid)trimethine oxonol (DiSBAC2(3)); CCCP, carbonylcyanide chlorophenylhydrazone.

(^2)
L. Vieira and Z. I. Cabantchik, unpublished observations.


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