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Originally published In Press as doi:10.1074/jbc.M404671200 on July 16, 2004

J. Biol. Chem., Vol. 279, Issue 38, 39438-39446, September 17, 2004
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Evidence for Activation of Endogenous Transporters in Xenopus laevis Oocytes Expressing the Plasmodium falciparum Chloroquine Resistance Transporter, PfCRT*

Susanne Nessler{ddagger}§, Oliver Friedrich{ddagger}§, Naziha Bakouh||**, Rainer H. A. Fink¶, Cecilia P. Sanchez{ddagger}, Gabrielle Planelles||, and Michael Lanzer{ddagger}{ddagger}{ddagger}

From the {ddagger}Hygiene Institut, Abteilung Parasitologie, Universitätsklinikum Heidelberg, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, the Medizinische Biophysik, Institut für Physiologie und Pathophysiologie, Universität Heidelberg, Im Neuenheimer Feld 326, D-69120 Heidelberg, Germany, and ||INSERM, Unité 467, Université Paris V, Faculté de Médecine Necker-Enfants Malades, 75730 Paris Cedex 15, France

Received for publication, April 27, 2004 , and in revised form, July 12, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A large body of genetic, reverse genetic, and epidemiological data has linked chloroquine-resistant malaria to polymorphisms within a gene termed pfcrt in the human malarial parasite Plasmodium falciparum. To investigate the biological function of the chloroquine resistance transporter, PfCRT, as well as its role in chloroquine resistance, we functionally expressed this protein in Xenopus laevis oocytes. Our data show that PfCRT-expressing oocytes exhibit a depolarized resting membrane potential and a higher intracellular pH compared with control oocytes. Pharmacological and electrophysiological studies link the higher intracellular pH to an enhanced amiloride-sensitive H+ extrusion and the low membrane potential to an activated nonselective cation conductance. The finding that both properties are independent of each other, together with the fact that they are endogenously present in X. laevis oocytes, supports a model in which PfCRT activates transport systems. Our data suggest that PfCRT plays a role as a direct or indirect activator or modulator of other transporters.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Malaria remains one of the most important infectious diseases, causing an estimated 300–500 million clinical cases and 1–3 million deaths annually (1). The current situation is largely due to the spread of chloroquine-resistant Plasmodium falciparum strains and a lack of alternative drugs that can replace chloroquine as a first-line antimalarial drug. Chloroquine is believed to exert its antimalarial activity by interfering with endogenous heme detoxification pathways (2). During intraerythrocytic development, P. falciparum degrades the host cell hemoglobin in its acidic food vacuole, releasing large quantities of heme, which are detoxified, presumably through peroxidative degradation (3), glutathione-dependent decomposition (4), and/or crystallization to inert hemozoin (5). Chloroquine prevents heme detoxification pathways by forming complexes with heme (6–9). The buildup of toxic membrane-associated heme-chloroquine complexes eventually destroys the integrity of the parasite's membranes (10–14). The consensus of evidence indicates that chloroquine-resistant strains control access of chloroquine to its intracellular binding site heme (15, 16), through an active drug efflux carrier system (17, 18), novel heme-binding proteins (15, 16), or changes in vacuolar pH, which would affect heme detoxification processes (19–21) or acidotropic drug accumulation (22, 23).

Chloroquine resistance has been linked to polymorphisms within a gene termed pfcrt (for P. falciparum chloroquine resistance transporter) residing on chromosome 7 of the human malarial parasite P. falciparum (24–27). pfcrt encodes a protein of 421 amino acids with 10 predicted membrane-spanning domains located in the membrane of the parasite's food vacuole (24). BLAST analysis has revealed no obvious homologies of PfCRT to proteins of known function. However, one study has reported a vague structural resemblance of PfCRT to aqueous chloride channels (28), whereas another study has suggested that PfCRT belongs to the drug/metabolite transporter family (29).

Thus far, only one study has attempted to investigate the function of PfCRT. Heterologous expression of PfCRT fusion proteins in Pichia pastoris and the subsequent investigation of transport properties displayed by inside-out membrane vesicles suggested a function for PfCRT in passive transmembrane Cl– movement (30). However, it is not clear from these data whether PfCRT itself mediates Cl– movement, e.g. by functioning as a chloride channel, or whether the observed effects are of a secondary nature due to the activation of endogenous channels. Thus, there is very little information available regarding the biological function of PfCRT during parasite development or regarding its role as a mediator of chloroquine resistance. A better understanding of the properties of PfCRT would be an important step toward novel intervention strategies, which may either circumvent the chloroquine resistance mechanism or inactivate it, thereby revitalizing chloroquine as a first-line drug.

In search of an alternative, more amenable heterologous expression system to study the function of PfCRT, we have expressed, in Xenopus laevis oocytes, the wild-type pfcrt allele from the chloroquine-sensitive P. falciparum clone HB3. Successful membrane expression of the protein was associated with changes in oocyte properties. Compared with control cells, PfCRT-expressing oocytes exhibited a more alkaline intracellular pH and a less negative transmembrane potential difference. The alkaline intracellular pH reverted to control values in the presence of Na+-H+ exchanger inhibitors. The membrane potential hyperpolarized upon removal of extracellular Na+ or in the presence of diphenylamine-2-carboxylic acid. We conclude from our data that PfCRT expression activates the endogenous Na+-H+ antiporter and an endogenous nonselective cation conductance, both present in the plasma membrane of X. laevis oocytes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—All chemicals were purchased from Sigma, except Hoechst 694 ((3-methyl-sulfonyl-4-piperidinobenzoyl)guanidine methanesulfonate), which was a gift from Dr. H. J. Lang (Aventis).

Biological Material—X. laevis adult females were purchased from CNRS (Montpellier, France) and NASCO and kept as described (31). Anesthesia was administered by immersion in iced water supplemented with 2 mM ethyl n-aminobenzoate methanesulfonate. Ovarian fragments were incubated in collagenase A-containing Ringer's solution as described previously (32). After rinsing, stage V–VI oocytes were defolliculated using fine forceps.

Expression of pfcrt cRNA in X. laevis Oocytes—To obtain PfCRT expression in X. laevis oocytes, the pfcrt allele of the chloroquine-sensitive P. falciparum clone HB3 was reconstructed on the basis of the yeast codon usage (Geneart, Regensburg, Germany). The resulting fragment was cloned into the BglII site of the X. laevis oocyte expression vector pSP64T (33, 34). The construct was designed such that the authentic protein was produced in X. laevis oocytes without any additional amino acids. The cRNA of PfCRT was prepared using the mMESSAGE mMACHINE kit (Ambion Inc.). cRNA was dissolved in RNase-free water, stored at –80 °C, and diluted to a concentration of 400 ng/µl immediately prior to injection. 50 nl (containing 20 ng of cRNA or water for the controls) were injected into defolliculated stage V–VI X. laevis oocytes using a Nanoject automatic injector (Drummond Science Inc., Broomall, PA).

Immunodetection of PfCRT—2–3 days after cRNA injection into oocytes, membranes were prepared from X. laevis oocytes (35) and solubilized in SDS-PAGE loading buffer. Membrane proteins were size-fractionated on a 12% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. PfCRT was detected using a guinea pig anti-peptide antiserum (1:1000 dilution) raised against the N terminus of PfCRT (MKF ASK KNN QKN SSK; Eurogentec). As a secondary antibody, we used a peroxidase-conjugated donkey anti-guinea pig antiserum (1:10,000 dilution) purchased from Dianova.

Immunofluorescence—2 days after cRNA injection, oocytes were embedded in Tissue-Tek cryo-embedding compound (Miles Inc.); frozen in liquid propane; which was cooled by liquid nitrogen; and stored at –80 °C until used. Sections (5 µm) were cut at –26 °C with a cryomicrotome and mounted on glass slides. For immunofluorescence staining, sections were fixed for 15 min with 4% paraformaldehyde in phosphate-buffered saline (PBS). PfCRT was detected using the guinea pig anti-PfCRT antiserum. As a secondary antibody, a Cy3-conjugated donkey anti-guinea pig antiserum (1:100 dilution; Dianova) was used. Images were taken with a Zeiss LSM 510 confocal laser scanning microscope.

Electrophysiological Approaches—For electrophysiological experiments, oocytes were placed in a Plexiglas microchamber and superfused by the gravimetric delivery of artificial solutions, differing from each other by a single parameter. The membrane potential (Vm) and the intracellular pH (pHi), intracellular Na+ activity ({alpha}Na), or intracellular Cl– activity ({alpha}Cl) were simultaneously measured using double-barreled ion-selective microelectrodes. The details of microelectrode manufacture have been described previously (32, 36). In this study, the tip of the pH-sensitive barrel was filled with Fluka H+ ionophore 95291 and its shank with 67 mM NaCl, 40 mM KH2PO4, and 23 mM NaOH. Na+- and Cl–-selective electrodes were filled with Fluka 71176 and Corning 477913, respectively, and the shank was backfilled with 100 mM NaCl. The conventional barrel contained 1 M KCl. When necessary, the double-barreled microelectrode was gently beveled prior to use on a microgrinder (de Marco Eng., Geneva, Switzerland). Double-barreled microelectrodes were placed on a three-dimensional micromanipulator (MM 33, Narishige, Tokyo, Japan) and connected via Ag/AgCl electrodes to the input of an ultrahigh impedance electrometer (WPI FD 223, Aston, United Kingdom). Before each experiment, the slope of the selective microelectrode was determined from the change in potential induced by a change in Cl– or Na+ concentration or in pH value of the superfusing solution. The slope was systematically checked again at the end of each experiment. The slope was 55–62 mV/pH unit for pH-sensitive microelectrodes, 50–59 mV/concentration decade change for Na+-sensitive microelectrodes, and 49–54 mV/concentration decade change for Cl–-sensitive microelectrodes. We verified that the slope was not affected by the different solutions used in this study. Upon impalement of an oocyte, the conventional barrel of the microelectrode measured the Vm, and the selective barrel measured the electrochemical potential of the ion under investigation (Vsel) across the cell membrane. The electrometer output displayed on a multichart recorder (Sefram, Servofram, Saint Etienne, France) gave both the Vm signal and the algebraic sum of Vsel – Vm.

The intracellular ionic activity of the ion i (Ai) was calculated using the following general relationship: Ai = Aref x 10(Vsel – Vm)/S, where Aref is the ionic activity for i in the reference solution (using an activity coefficient of 0.75) and S is the slope. For experiments with pH-sensitive microelectrodes, this general equation reduces to the simplified relationship pHi = pHref – (VH – Vm)/S, where pHref is the pH of the Ringer's solution, and VH is the proton electrochemical potential across the cell membrane. In experiments in which only Vm was needed, conventional microelectrodes filled with 1 or 3 M KCl were used. Preliminary experiments showed that the measured Vm was similar when using 1 or 3 M KCl to backfill conventional microelectrodes. The electrical circuit was closed by a KCl (1 or 3 M)-agarose Ag/AgCl macroelectrode placed in the bath.

To obtain oocyte-specific membrane conductances (Gm) and current-voltage relationships, cells were punctured with two separate microelectrodes filled with 3 M KCl (resistance of <5 megaohms). Experiments were controlled using a GeneClamp 500 amplifier (Axon Instruments, Inc., Foster City, CA) (37, 38). To avoid bath error potentials induced by superfusate changes, the bath potential was virtually clamped to 0 mV using a two-electrode virtual ground circuit (37). Electrode tip potentials were adjusted to 0 mV in the bulk solution prior to penetration of the oocytes. Membrane capacitance was compensated for by ~90% using the capacitance compensation circuit of the amplifier. To account for resistance errors due to electrode tip polarizations, the tip potential was measured at the end of an experiment after electrode withdrawal from the oocyte, and experiments during which the tip potentials varied by >2 mV were discarded. The Gm was obtained from current clamp experiments as follows. The Vm was set to –70 mV by injection of a constant current from which current pulse steps ranging from –10 to +30 nA in 10-nA increments were applied for 8–10 s, and the change in Vm was recorded. The input conductance was calculated from the slope of the linear steady-state Vm-current relationship. Gm was determined by relating the input conductance to the membrane surface of the oocytes, calculated after optical measurement of the oocyte diameter.

For voltage clamp recordings, the holding potential was adjusted to –70 mV, and potential step pulses lasting for 15 s were applied from –120 to +20 mV in 20-mV increments while the membrane current (Im) was recorded. The current-voltage relationship was determined from the steady-state Im values.

Artificial Solutions—Prior to the experiments, oocytes were kept at 18 °C in an amphibian-adapted Ringer's solution (96 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES adjusted to pH 7.5 with NaOH) supplemented with penicillin/streptomycin. Experiments were performed at room temperature. In Na+-free solutions, NaCl was replaced with an equimolar amount of choline chloride or lithium chloride, and the pH was adjusted using Trizma (Tris base). In the Cl–-free solutions, chloride salts were replaced with gluconate salts, and the calcium concentration was raised 3-fold to compensate for calcium chelation by gluconate–. When using -buffered Ringer's solution, a 21 mM concentration of the bulk of NaCl was replaced with an equimolar concentration of , and the solution was equilibrated with CO2 at a pressure of 3 kilopascals to pH 7.5. In ammonium-containing solutions, 20 mM NH4Cl were used at the expense of NaCl. In cell acid loading experiments, a 40 mM sodium salicylate-containing solution (NaCl was reduced by an equivalent amount) was used. Where indicated, the Ringer's solution was supplemented with diphenylamine-2-carboxylic acid (DPC1; 1 mM), ouabain (0.1 mM), amiloride (1 or 0.1 mM), Hoechst 694 (50 µM), or ethylisopropylamiloride (EIPA; 50 µM).

Statistics—Results are expressed as means ± S.E., with n = number of oocytes investigated from at least three independent experiments. Significance of the results was assessed by paired or unpaired Student's t test, and results were considered significantly different for p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of PfCRT in X. laevis Oocytes—Translation of pfcrt cRNA in Xenopus oocytes was verified by Western analysis using a guinea pig antiserum specific to PfCRT, which identified a protein with the expected size of 48.6 kDa (Fig. 1a). The bulk of PfCRT was found associated with the total membrane fraction (Fig. 1a). Immunofluorescence revealed co-localization of PfCRT over the oolemma. In addition, a dotted staining pattern was observed within the yolk (Fig. 1b).



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  		FIG. 1.
Expression of pfcrt in Xenopus oocytes. a, cytosolic extracts and membrane preparations from control oocytes (–) and PfCRT-expressing oocytes were prepared and size-fractionated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and analyzed using a guinea pig anti-peptide antiserum raised against the N terminus of PfCRT. As a positive control, a total protein extract from P. falciparum strain HB3 was analyzed in parallel. Size standards are indicated in kilodaltons. b, shown is the immunofluorescence of PfCRT in Xenopus oocytes. Upper panel, cryosections were stained with the anti-PfCRT antiserum. As a secondary antibody, the Cy3-conjugated donkey anti-guinea pig antiserum was used. Lower panel, shown is a phase-contrast image. Bar = 20 µm.

 
PfCRT Expression Alters Oocyte pHi and Vm—To determine whether PfCRT expression modifies basic properties of the oocytes, we measured the resting Vm, pHi, {alpha}Na, and {alpha}Cl 2 days after cRNA injection. Water-injected oocytes were measured in parallel as controls. Compared with control oocytes, for which measured values were consistent with published data (32, 39), PfCRT-expressing oocytes exhibited a higher pHi and a lower Vm (Table I). {alpha}Na and {alpha}Cl were similar in control and PfCRT-expressing oocytes (Table I). Using a -instead of a HEPES-buffered Ringer's solution had no effect on the Vm values (data not shown), yet slightly reduced pHi while maintaining the pHi differences observed between PfCRT-expressing and control oocytes (Table I). This finding suggests that the effects of pfcrt expression on the oocyte Vm and pHi are -independent. For this reason, we performed further investigations in a nominal -free buffer.


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  		TABLE I
Vm, pHi, amiloride-sensitive pHi recovery rate from an acid load ({Delta}pHi/{Delta}t), {alpha}Na, and {alpha}Cl of control and PfCRT-expressing oocytes

The mean ± S.E. of n independent determinations is shown. The resting pHi values given were determined in HEPES-buffered Ringer's solution. In -buffered Ringer's solution, the resting pHi values were as follows: 7.20 ± 0.02 (n = 3) for control oocytes and 7.46 ± 0.01 (n = 3) for PfCRT-expressing oocytes. NS, not significant.

 
To better define the temporal changes in Vm and pHi, we measured both parameters every 6 h following the injection of the pfcrt cRNA. Both the Vm and pHi began to diverge from the control values, determined in mock-transfected oocytes, ~16 h after cRNA injection and approached steady-state values after 42 h (Fig. 2, a and b). Subsequent experiments were performed 46–72 h after cRNA (or water) injection.



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  		FIG. 2.
Temporal changes in basic properties of X. laevis oocytes expressing PfCRT. Temporal changes in the resting Vm (a) and pHi (b) were determined in control ({circ}) and PfCRT-expressing () oocytes following injection of cRNA or water (in the case of control oocytes). The means ± S.E. of six independent determinations are shown.

 
PfCRT Expression Enhances Proton-equivalent Extrusion—We next sought to investigate the cause of the higher resting pHi observed in PfCRT-expressing oocytes compared with control oocytes. Previous studies have shown that the main pHi regulatory mechanism in oocytes is Na+-H+ exchange (39). We therefore reasoned that altered Na+-H+ exchanger (NHE) activity may be responsible for the higher pHi observed in PfCRT-expressing oocytes. To verify this hypothesis, we incubated oocytes in the presence of amiloride (1 and 0.1 mM), Hoechst 694 (50 µM), and EIPA (50 µM). These compounds are established inhibitors of NHEs (40), including that of X. laevis oocytes (39, 41). Although no immediate changes in pHi were observed, incubations for 4 h in the presence of NHE inhibitors affected the pHi in both control and PfCRT-expressing oocytes (Fig. 3a). However, the observed intracellular acidification was significantly stronger in PfCRT-expressing oocytes, with pHi values now approaching those of control oocytes (Fig. 3a). Incubating oocytes in the presence of amiloride or other NHE inhibitors for 4 h did not alter the Vm in either control or PfCRT-expressing oocytes (data not shown). These data are consistent with amiloride and the other compounds inhibiting an electroneutral H+ extrusion system, viz. NHE.



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  		FIG. 3.
Effect of NHE inhibitors on the resting pHi and pHi recovery after an acid load. a, control (open bars) and PfCRT-expressing (closed bars) oocytes were incubated for 4 h in the presence of amiloride (AM; 1.0 and 0.1 mM), Hoechst 694 (Hoe 694; 50 µM), and EIPA (50 µM) before the pHi was determined. The experiments were conducted 2 days after pfcrt cRNA injection. b, shown are the {Delta}pHi values during the first 10 min after acid loading, brought about by temporally superfusing oocytes in a salicylate-containing solution, in control (open bars) and PfCRT-expressing (closed bars) oocytes. pH recovery after acidification was monitored in the presence and absence of amiloride (1.0 mM). In both a and b, the means ± S.E. of three or more independent determinations are shown. *, significant differences in the pHi or {Delta}pHi (p < 0.05) in control and PfCRT-expressing oocytes; #, significant differences in the pHi or {Delta}pHi (p < 0.01) in the absence and presence of NHE inhibitors in PfCRT-expressing oocytes.

 
To verify the conclusion that PfCRT-expressing oocytes have an activated NHE, we determined the rate of pHi recovery after an acid load, brought about by superfusing the oocytes in a salicylate-containing solution. Exposure to the salicylate-containing solution acidified the cells to similar pHi values (7.03 ± 0.03 (n = 6) and 6.97 ± 0.02 (n = 6) for PfCRT-expressing and control oocytes, respectively). Upon salicylate withdrawal, the pHi recovery rates were then determined in the presence and absence of amiloride in a paired fashion. As shown in Fig. 3b, in the absence of amiloride, recovery of the pHi to its resting value was faster in PfCRT-expressing oocytes than in control oocytes. The amiloride-sensitive component of the pHi recovery rates was (1.27 ± 0.2) x 10–2 pH units/min (n = 3) for PfCRT-expressing oocytes and (0.24 ± 0.15) x 10–2 pH units/min (n = 3) for control oocytes (p < 0.001) (Table I).

The results suggest that, in PfCRT-expressing oocytes, the alkaline resting pHi is due to an increase in H+ extrusion mediated by an amiloride-sensitive NHE. Moreover, the high resting pHi is apparently not directly related to the low Vm in PfCRT-expressing oocytes.

PfCRT Expression Activates a Nonselective Cation Conductance—To investigate the low Vm values observed in PfCRT-expressing oocytes, we first determined the Gm in control and PfCRT-expressing oocytes. From an initial holding Vm of –70 mV, step currents of –10, +10, +20, and +30 nA were applied to the oocytes, and temporal changes in Vm were monitored (Fig. 4a). A significantly higher Gm value of 48.10 ± 6.7 microsiemens/cm2 (n = 24) was observed in PfCRT-expressing oocytes compared with control oocytes, which had a Gm of 15.09 ± 0.97 microsiemens/cm2 (n = 23; p < 0.001) (Fig. 4b). Removing Na+ (substituted with an equimolar amount of choline+) or Cl– (substituted with an equimolar amount of gluconate–) from the bath had virtually no effect on the Gm in control oocytes, but substantially reduced the Gm in PfCRT-expressing oocytes (Fig. 4b).



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  		FIG. 4.
Gm in control and PfCRT-expressing oocytes. The Gm was determined in control and PfCRT-expressing oocytes bathed in Ringer's solution, a Cl–-free solution, and an Na+-free solution. The Vm was initially adjusted to –70 mV before step currents were injected, and the temporal changes in Vm were monitored. a, original tracing of temporal changes in Vm in response to the step currents recorded from a PfCRT-expressing oocyte. The inset details the experimental protocol. The superfusates used were Ringer's solution (dotted line), a Cl–-free solution (dashed line), and an Na+-free solution (solid line). b, statistical analysis of the data obtained. The means ± S.E. of 10 or more independent determinations are shown. Open bars, control oocytes; closed bars, PfCRT-expressing oocytes; *, significant differences in the Gm (p < 0.001) in control and PfCRT-expressing oocytes; µS, microsiemens.

 
To further characterize the enhanced Gm observed in PfCRT-expressing oocytes, current-voltage relationships were established over a range from –120 to +20 mV. Fig. 5a shows representative recordings of membrane currents of oocytes bathed in Ringer's solution. Consistent with measured steady-state Vm values (zero current), the current-voltage relationship of PfCRT-expressing oocytes was shifted by about +20 mV compared with that of control cells (Fig. 5b). Moreover, a slowly activating inward rectification was observed at negative voltage (Fig. 5b). Cl– removal had little effect on the current-voltage relationships of control oocytes (Fig. 5b). In PfCRT-expressing oocytes, however, Cl– removal considerably reduced the inward rectification observed at negative voltage without significantly affecting the potential at which currents inverted polarity (Erev). In comparison, Na+ removal shifted the Erev to a more negative value (from –19.99 ± 0.83 to –34.23 ± 1.86 mV) (Fig. 5c). In the range of Erev (–40 to –20 mV), Na+ removal also induced a reduction in Gm (from 36.04 ± 2.87 microsiemens/cm2 (n = 40) to 23.12 ± 2.73 microsiemens/cm2 (n = 11; p < 0.01)) in PfCRT-expressing oocytes as determined from the current-voltage relationships (Fig. 5c). At Vm values more negative than –40 mV, the reduction in Gm was even more pronounced, consistent with the data shown in Fig. 4. Na+ removal had no significant effect on control oocytes (Fig. 5c). These findings suggest that, in PfCRT-expressing oocytes, the low resting Vm is associated with an increased Gm and a change in ion selectivity of the membrane, which, in turn, is consistent with the activation of a cation conductance(s).



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  		FIG. 5.
Steady-state current-voltage relationship of control and PfCRT-expressing oocytes. a, representative recordings of the Im observed in control (left panel) and PfCRT-expressing (right panel) oocytes. The cells were bathed in Ringer's solution. The pulse protocol is shown in the inset. b, current-voltage relationships of control ({triangleup} and {blacktriangleup}) and PfCRT-expressing ({circ} and ) oocytes determined in Ringer's solution (closed symbols) and a Cl–-free solution (open symbols). c, current-voltage relationships of control ({triangleup} and {blacktriangleup}) and PfCRT-expressing ({circ} and ) oocytes determined in Ringer's solution (closed symbols) and an Na+-free solution (open symbols). The data represent the means ± S.E. of nine or more independent determinations. In some cases, the error bars are smaller than the symbols and thus are not visible in the graphs.

 
To determine the nature of this conductance, the effects of extracellular ion substitutions on the Vm were monitored. Consistent with results from the previous protocol (zero current conditions), changing the superfusate from Ringer's solution to a Cl–-free solution did not significantly alter the Vm in both control and PfCRT-expressing oocytes (Fig. 6a and Table II). (The slight change in Vm observed was due to the liquid junction potential.) This finding suggests that the activation of a Cl– conductance is not predominantly responsible for the altered resting Vm observed in PfCRT-expressing oocytes. Switching the superfusate from Ringer's solution to an Na+-free solution had no major effect on the Vm in control oocytes. In the case of PfCRT-expressing oocytes, however, this experimental maneuver resulted in a dramatic membrane hyperpolarization, with the Vm now approaching the values measured in control oocytes (Fig. 6b and Table II). These data, together with the results obtained under voltage clamp conditions, strongly support the idea that PfCRT expression activates an Na+ or nonselective cation conductance (Gcat) in X. laevis oocytes.



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  		FIG. 6.
Representative recordings of the resting Vm and pHi in control and PfCRT-expressing oocytes determined under different extracellular bath conditions. a, effect of Cl– removal from the bath on the Vm and pHi. Arrows indicate the time point at which the superfusate was changed from Ringer's solution to a Cl–-free solution. b, effect of Na+ removal from the bath on the Vm and pHi. Arrows indicate the time point at which Na+ was removed (downward arrows) or reintroduced (upward arrows). c, effect of Na+ substitution with Li+ on the Vm. The downward arrow indicates the time point at which the superfusate was changed from Ringer's solution to an Na+-free solution. The upward arrow indicates the time point at which the superfusate was changed from an Na+-free solution to an Na+-free, Li+-containing solution. The dashed arrow indicates the change to Ringer's solution. d, effect of DPC on the Vm and pHi. Arrows indicate the time point at which 1 mM DPC was added to (downward arrows) or removed from (upward arrows) Ringer's solution. Solid lines, PfCRT-expressing oocytes; dashed lines, control oocytes. Bar = 2 min. Vm determinations were independent of whether a conventional microelectrode or a pH-sensitive double-barreled electrode was used. The statistical analysis of the data are compiled in Table II.

 


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  		TABLE II
Vm of control and PfCRT-expressing oocytes under different experimental conditions

The mean ± S.E. of n independent paired measurements is shown. NS, not significant.

 
The change in Vm induced by the Na+-free solution in PfCRT-expressing oocytes was fully reversible when the superfusate was switched back to an Na+-containing solution or an Na+-free solution containing Li+ as a substitute (Fig. 6, b and c; and Table II). In both cases, the Vm returned to its original value measured in Ringer's solution superfusate (Fig. 6, b and c; and Table II). This finding suggests that the conductance poorly discriminates between Na+ and Li+. The data are consistent with the activation of a Gcat rather than an Na+ conductance.

Previous studies have shown that Gcat can be inhibited by DPC in X. laevis oocytes (32, 42, 43). Although adding 1 mM DPC to the superfusate had only a marginal effect on the Vm in control oocytes, a strong membrane polarization was observed in the presence of DPC in PfCRT-expressing oocytes (Fig. 6d and Table II). This result is consistent with the activation of a DPC-sensitive Gcat in PfCRT-expressing oocytes. To further confirm this conclusion, we measured the changes in Vm ({Delta}Vm) induced by an increase in bath K+ concentration (]K+]+]o affects the equilibrium potentials of K+ and of the combined cationic species (Ecat). Experiments were performed in the presence of ouabain (0.1 mM) to inhibit the endogenous Na+,K+ pump activity, which is known to be sensitive to [K+]o (32), and under Na+-free conditions to remove the major substrate of Gcat. As shown in Fig. 7, under such experimental conditions, a 5-fold increase in [K+]o induced a significant membrane depolarization in control oocytes, which was insensitive to DPC. These findings suggest that, in control oocytes, the K+-induced membrane depolarization results from a membrane partial conductance to K+ (GK) rather than from a Gcat. In comparison with control oocytes, the high K+ solution induced a significantly lower {Delta}Vm in PfCRT-expressing oocytes, which may indicate a reduced GK and/or an enhanced Gcat. The latter assumption was supported by the significant increase in high K+-induced {Delta}Vm in the presence of DPC. As the Ecat was shifted to negative values under the Na+-free conditions used in this experiment, the {Delta}Vm increased in the presence of DPC if the Gcat was enhanced. Thus, the lower {Delta}Vm induced by high K+ in PfCRT-expressing oocytes compared with that in control oocytes reveals the presence of an enhanced Gcat, in addition to a GK.



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  		FIG. 7.
Changes in Vm induced by increasing the [K+]o in control and PfCRT-expressing oocytes. The extracellular K+ concentration was increased 5-fold, and {Delta}Vm values were monitored. The experiment was performed in the presence (shaded bars) and absence (open bars) of DPC. The bath contained ouabain (0.1 mM) and was Na+-free. The data represent the means ± S.E. of four or more independent determinations. *, significant differences in {Delta}Vm (p < 0.05) in control oocytes and PfCRT-expressing oocytes.

 
Previous studies have shown that the Gcat allows for an increased NH+4 influx in oocytes (31, 32). We therefore investigated the effect of 20 mM NH4Cl on the pHi. A significantly faster NH4Cl-induced intracellular acidification (expressed as pHi change as a function of time, {Delta}pHi/{Delta}t) was observed in PfCRT-expressing oocytes compared with control oocytes ({Delta}pHi/{Delta}t = 0.22 ± 0.02 (n = 17) versus {Delta}pHi/{Delta}t = 0.08 ± 0.01 (n = 27); p < 0.001). The faster cell acidification measured in PfCRT-expressing oocytes is consistent with an accelerated NH+4 influx, followed by the partial intracellular dissociation of NH+4 into NH3 and H+ (31, 44). Taken together, our data provide strong evidence for activation of the Gcat in PfCRT-expressing X. laevis oocytes.

To investigate whether the lower Vm and the more alkaline pHi observed in PfCRT-expressing oocytes are related, we measured the effect of external ion substitutions on the pHi. As shown in Fig. 6, the pHi was not affected by Na+ or Cl– removal from the bath or by the addition of DPC, at least within the timeframe measured (15 min). These data support the view that the changes in Vm and pHi observed in PfCRT-expressing oocytes are two independent events.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here, we have reported the successful expression of the P. falciparum chloroquine resistance transporter, PfCRT, in X. laevis oocytes. Expression was not possible until we reconstructed the coding sequence on the basis of the yeast codon usage. The high A/T content of the original P. falciparum sequence most likely prevented efficient translation, a problem often encountered when working with DNA from this parasite. The reconstructed pfcrt coding sequence was designed such that translation of the corresponding in vitro generated cRNA would produce the original full-length protein in X. laevis oocytes without generating any kind of fusion protein. Successful expression was confirmed by Western analysis, and immunofluorescence located the protein at the oocyte plasma membrane (Fig. 1).

PfCRT-expressing oocytes revealed changes in two basic physiological parameters. They exhibited a low resting Vm and an alkaline pHi. In addition, a chloride-dependent inward rectification was observed, which we did not explore further since it was apparent only at an unphysiologically negative Vm. At first sight, the modifications seen in PfCRT-expressing oocytes are in agreement with studies postulating a function for PfCRT in ion transport mechanisms or pH regulation (21, 28, 30, 45). For example, it has been suggested that PfCRT mediates active or passive transmembrane Cl– movement (28, 30, 45), and another study has correlated polymorphisms within PfCRT associated with chloroquine resistance with a reduced food vacuolar pH in P. falciparum (45).

However, upon investigating the mechanisms underpinning the alkaline pHi and the low Vm observed in PfCRT-expressing oocytes, we were drawn to a different conclusion. Concerning the alkaline pHi, we consider it unlikely that PfCRT itself mediates amiloride-sensitive H+ extrusion. Instead, we favor the hypothesis that the alkaline pHi observed in PfCRT-expressing oocytes is related to the activation of an endogenous NHE. We first noted that, from a thermodynamic point of view, the transmembrane chemical Na+ gradient is large enough to drive the pHi to the values recorded in PfCRT-expressing cells. A more direct argument in favor of activated Na+-H+ exchange is that established NHE inhibitors (40), such as amiloride, EIPA, and Hoechst 694, acidify the pHi in PfCRT-expressing oocytes to values similar to those measured in control oocytes under the same experimental conditions (Fig. 3a). Amiloride, EIPA, and Hoechst 694 are also known to inhibit the NHE of X. laevis oocytes (39, 41). Further supporting the activation of an NHE, the amiloride-sensitive pHi recovery rate from an acid load was significantly higher in PfCRT-expressing oocytes than in control oocytes (Fig. 3b and Table I). PfCRT has no homologies to NHEs or H+-ATPases and lacks an amiloride-binding site (46).

Concerning the low Vm measured in PfCRT-expressing oocytes, several lines of evidence suggest that the underpinning mechanism is a Gcat induced by PfCRT expression. First, under voltage clamp conditions, when PfCRT-expressing oocytes were superfused with an Na+-free (substituted with choline+) medium, the Gm decreased, and the Erev shifted to more negative values (Fig. 5c). Replacing Na+ with choline+ induced a huge membrane hyperpolarization, which was not observed when Li+ was used instead of choline+ (Fig. 6, b and c). These results are consistent with an ion conductance having a poor discrimination between Na+ and Li+. Second, DPC, an established inhibitor of the oocyte Gcat (32), induced a strong Vm hyperpolarization (Fig. 6d). This finding is in accordance with the inhibition of a DPC-sensitive conductance. Third, as expected from a Gcat, discrimination between Na+ and K+ was poor. The depolarization induced by a high K+ solution was significantly lower in PfCRT-expressing oocytes than in control oocytes. Moreover, in PfCRT-expressing oocytes, but not in control cells, the K+-induced depolarization increased in the presence of DPC (Fig. 7). Finally, NH+4 influx seems to be enhanced in PfCRT-expressing oocytes. This again points toward a Gcat, which, according to previous reports, is permeable for NH+4 in X. laevis oocytes (31, 44).

Although our data demonstrate an enhanced amiloride-sensitive H+ extrusion and the presence of a pathway permeable to cationic species in PfCRT-expressing oocytes, we do not suggest that PfCRT itself mediates these ion transport properties. A direct role of PfCRT in H+ extrusion and in nonselective cation transport is unlikely since these transmembrane pathways appear to be independent of each other both electrophysiologically and pharmacologically. For example, some external ion replacements induced rapid changes in the Vm without inducing concurrent changes in the pHi (Fig. 6). Given that both the Gcat and NHE are present in the plasma membrane of X. laevis oocytes (39, 41, 47), the simplest explanation is that the low Vm and the high pHi observed in PfCRT-expressing oocytes result from the activation of endogenous transport systems, viz. an NHE and a Gcat. Despite their enhanced activities, no significant differences in {alpha}Na between PfCRT-expressing and control oocytes were measured (Table I). A plausible explanation is that the cells have adjusted Na+ efflux, mediated by the Na+,K+-ATPase, to maintain {alpha}Na. It is well known that the Na+,K+-ATPase activity, in X. laevis oocytes as well as in other cells, is highly sensitive to changes in {alpha}Na (48, 49). The activation of an NHE and a Gcat appears to be a specific effect, as expressing conducting and non-conducting heterologous proteins does not result per se in the activation of endogenous transporters in X. laevis oocytes (50–52).

A previous study has suggested that PfCRT mediates transmembrane Cl– movement (30). This conclusion is based on the observation that inside-out vesicles from P. pastoris membranes containing PfCRT fused to a biotin acceptor domain acidified only in the presence of a symmetrical transmembrane Cl– gradient. Acidification consumed ATP and was more pronounced in vesicles containing the mutant PfCRT protein associated with chloroquine resistance than in those containing the wild-type protein. As PfCRT itself has no homologies to H+-ATPases, it was argued that PfCRT supports endogenous H+-ATPase activity to produce larger {Delta}pH values by facilitating transmembrane Cl– movement, which would shunt changes in Vm resulting from the H+-ATPase activity (30). Is it possible that, in X. laevis oocytes, PfCRT induces changes in Cl– homeostasis that subsequently activate endogenous transport systems? Although this is an intriguing hypothesis, our data provide no supporting evidence. The Vm, Erev, and pHi did not respond to external Cl– removal in PfCRT-expressing oocytes, and no significant differences in {alpha}Cl were observed between control and PfCRT-expressing oocytes.

Although we found no evidence for a direct or indirect function of PfCRT in Cl– conductance, the similarities between the two studies are nevertheless striking. In both cases, the expression of PfCRT resulted in the activation of endogenous transport systems: the Gcat and NHE in X. laevis oocytes and an H+-ATPase in P. pastoris. One may wonder whether the Cl– movement observed in the P. pastoris system is also due to the activation of an endogenous Cl– transporter by PfCRT.

Thus, there appears to be a common theme of PfCRT activating or modulating transport systems. Currently, we have no explanation of the underlying mechanism. It may involve protein-protein interactions, as exemplified by the activation of an Na+-K+-2Cl– cotransporter by the trout erythrocyte anion exchanger-1 (53) or of channels by single membrane-spanning proteins (54–59). It may also involve electric or thermodynamic coupling such as with the Na+-K+-2Cl– cotransporter and KCl transporter (60). As reported for amino acid transporters, coupling of transport activities may serve to recycle ions or to buffer ionic or Vm changes induced by the transporter of interest (61–64). Alternatively, PfCRT may interfere with second messengers, such as Ca2+, which is known to regulate the activity of numerous membrane transporters, including the NHE (46, 65), Gcat (66), and H+-ATPase (67).

A function for PfCRT as an activator or modulator of other transporters would be consistent with most models proposed to explain chloroquine resistance in P. falciparum. Mutations within PfCRT that create chloroquine resistance may alter functional coupling of PfCRT to other transport systems, as seen in the P. pastoris system (30), which, in turn, may affect chloroquine transport or pH regulation of the food vacuole. Further studies will help distinguish between these possibilities.


   FOOTNOTES
 
* This work was supported in part by the Landesstiftung Baden-Württemberg. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

** Supported by a grant from the Fondation de la Recherche Médicale. Back

{ddagger}{ddagger} To whom correspondence should be addressed. Tel.: 49-6221-567-845; Fax: 49-6221-564-643; E-mail: michael_lanzer{at}med.uni-heidelberg.de.

1 The abbreviations used are: DPC, diphenylamine-2-carboxylic acid; EIPA, ethylisopropylamiloride; NHE, Na+-H+ exchanger. Back


   ACKNOWLEDGMENTS
 
We thank G. Krohne for helping with immunofluorescence, C. A. Karle for helping with oocyte transfection, H. J. Lang for Hoechst 694, and S. Krishna for pSP64T.



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