Differences in trans-stimulated chloroquine efflux kinetics arelinked to PfCRT in Plasmodium falciparum

Cecilia P. Sanchez1, Petra Rohrbach1, Jeremy E. McLean1, David A. Fidock2, Wilfred D.Stein3, and Michael Lanzer1,*1 Hygiene Institut, Abteilung Parasitologie, Universitätsklinikum Heidelberg, Im Neuenheimer Feld324, 69120 Heidelberg, Germany2 Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY10461, USA3 Biological Chemistry, Silberman Institute of Life Sciences, Hebrew University of Jerusalem, GivatRam, Jerusalem 91904, Israel

SummaryThe mechanism underpinning chloroquine drug resistance in the human malarial parasitePlasmodium falciparum has remained controversial. Currently discussed models include a carrier ora channel for chloroquine, the former actively expelling the drug, the latter facilitating its passivediffusion, out of the parasite’s food vacuole, where chloroquine accumulates and inhibits haemdetoxification. Here we have challenged both models using an established trans-stimulation effluxprotocol. While carriers may demonstrate trans-stimulation, channels do not. Our data reveal thatextracellular chloroquine stimulates chloroquine efflux in the presence and absence of metabolicenergy in both chloroquine-sensitive and -resistant parasites, resulting in a hyperbolic increase in theapparent initial efflux rates as the concentration of external chloroquine increases. In the absence ofmetabolic energy, the apparent initial efflux rates were comparable in both parasites. Significantdifferences were only observed in the presence of metabolic energy, where consistently higherapparent initial efflux rates were found in chloroquine-resistant parasites. As trans-stimulation ischaracteristic of a carrier, and not a channel, we interpret our data in favour of a carrier for chloroquinebeing present in both chloroquine-sensitive and -resistant parasites, however, with different transportmodalities.

IntroductionFollowing the introduction of chloroquine as a first-line antimalarial drug in the late 1950s,malaria-related mortality among children declined by almost 50% in endemic areas (Carterand Mendis, 2002). The ground gained in the campaign against malaria was all but lost whenchloroquine resistance emerged in the field. Today, chloroquine-resistant (CQR) Plasmodiumfalciparum parasites are widespread. Due to a paucity of suitable replacement drugs, malariahas increased to an unprecedented 515 million clinical cases and more than one million deathsannually (Carter and Mendis, 2002; Snow et al., 2005).

*For correspondence. michael_lanzer@med.uni-heidelberg.de; Tel. (+49) 6221 567845; Fax (+49) 6221 564643.This material is available as part of the online article from http://www.blackwell-synergy.comPlease note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by theauthors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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Published in final edited form as:Mol Microbiol. 2007 April ; 64(2): 407–420. doi:10.1111/j.1365-2958.2007.05664.x.

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In its mode of action, chloroquine exploits a unique biochemical pathway of the parasite.During intraerythrocytic development, P. falciparum degrades haemoglobin of the hosterythrocyte to meet its nutrient requirements and to counteract osmotic imbalances resultingfrom its own anabolic activity (Lew et al., 2003). The haem released in this process ismineralized to inert hemozoin in the parasite’s acidic food vacuole (Egan et al., 2002; Fitch,2004). Chloroquine, which accumulates in this organelle (Yayon et al., 1984; Fitch, 2004), isthought to prevent haem bio-mineralization (Chou et al., 1980; Yayon et al., 1984; Dorn etal., 1998; Leed et al., 2002; De Dios et al., 2003; Fitch, 2004) by forming cytotoxic complexeswith haem that are lipophilic and that eventually kill the parasite by perforating cellularmembranes (Fitch et al., 1982; 1983). CQR P. falciparum parasites accumulate substantiallyless chloroquine (Fitch, 1970; 1973), apparently keeping their intracellular chloroquineconcentration below toxic levels.

The consensus of evidence indicates that chloroquine resistance is linked with polymorphismswithin the food vacuolar membrane protein PfCRT (Fidock et al., 2000; Djimde et al., 2001;Cooper et al., 2002; Sidhu et al., 2002; Lakshmanan et al., 2005). How PfCRT mediatesreduced chloroquine accumulation and, hence, the chloroquine resistance phenotype hasremained controversial. PfCRT groups with the drug/metabolite transporter superfamily(Martin and Kirk, 2004; Tran and Saier, 2004), which includes carriers that are capable oftransporting weak bases and/or divalent organic cations in co- or counter-transport.Accordingly, it has been suggested that the PfCRT associated with chloroquine resistance actsas an active efflux carrier of chloroquine (Fidock et al., 2000; Cooper et al., 2002; Zhang etal., 2004; Naude et al., 2005; Sanchez et al., 2005). This concept is consistent with trans-stimulation kinetics, which showed that pre-loaded intracellular chloroquine stimulated themaximal level of chloroquine uptake from the external medium in CQR parasites (Sanchez etal., 2003; 2005). Stimulated chloroquine uptake was inhibited by verapamil (Sanchez et al.,2004), and was causatively linked to the PfCRT polymorphisms associated with chloroquineresistance (Sanchez et al., 2005).

Contrasting with the proposed carrier model, other studies have suggested that the CQR PfCRTprotein acts as a voltage-gated channel facilitating the passive diffusion of protonatedchloroquine down its concentration gradient and out of the food vacuole (Warhurst et al.,2002; Zhang et al., 2004; Bray et al., 2006). The channel model was evoked to explain whyenergy deprivation or uncoupling the food vacuolar proton gradient had no effect on the rateof chloroquine efflux from CQR parasites (Bray et al., 2006). In all cases, chloroquine effluxwas rapid.

Consistent with both a carrier and a channel model, a recent study has shown that Dictyosteliumdiscoideum, expressing CQR PfCRT in acidic vesicles, exhibited reduced chloroquineaccumulation in a verapamil-sensitive manner, as compared with untransfected controls orslime moulds expressing the wild-type pfcrt allele (Naude et al., 2005). There is furtherevidence for chloroquine binding to PfCRT (Zhang et al., 2004) and PfCRT was shown toaffect chloroquine accumulation in reconstituted proteoliposomes (Tan et al., 2006). Todifferentiate between the carrier and the channel model, we have now investigated the kineticsof chloroquine efflux under ‘reverse varying-trans’ conditions (Stein, 1986). This procedureinvestigates whether extracellular unlabelled chloroquine would stimulate the release of pre-loaded labelled chloroquine. If chloroquine moves across a membrane via a carrier, one wouldexpect trans-stimulated chloroquine efflux, i.e. trans chloroquine should increase the apparentinitial efflux rate (Stein, 1986). Facilitated diffusion through a conventional pore or channeldoes not demonstrate trans-stimulation, because movements in different directions within apore or channel block rather than enhance one another (Keynes and Lewis, 1950; Dani andLevitt, 1981; Stein, 1986). Here we demonstrate trans-stimulated chloroquine efflux in CQRparasites in the presence and absence of metabolic energy. Unexpectedly, trans-stimulated

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chloroquine efflux was also observed in chloroquine-sensitive (CQS) parasites, but withkinetics differing from that of CQR parasites. These data suggest that both CQS and CQRparasites possess a carrier of chloroquine, however, with different transport modalities.

ResultsDifferences in chloroquine efflux kinetics between CQS and CQR parasites

Figure 1A depicts the time-courses of pre-loaded [3H]-chloroquine efflux from the CQS P.falciparum clone HB3 (chloroquine IC50 = 16.0 ± 1.0 nM) (closed symbols in this and allsubsequent figures) and the CQR clone Dd2 (chloroquine IC50 = 137 ± 11.0 nM) (open symbolsin this and all subsequent figures), over 10 min. The y-axis in this and the subsequent similarfigures represents the amount of labelled chloroquine that is still remaining within the parasitesat the time indicated on the x-axis. To account for the different chloroquine accumulation ratiosof the two parasites, HB3 and Dd2 were incubated with different concentrations of labelledchloroquine (20 nM and 100 nM respectively). These concentrations were empiricallydetermined to ensure comparable intracellular drug concentrations at the end of the pre-loadingphase (data not shown). The labelled chloroquine was rapidly released from the CQR parasite,but slowly from the CQS parasite, with rates of 0.55 ± 0.06 min−1 and 0.09 ± 0.04 min−1

respectively (Table 1). This observation confirms the original findings of Krogstad et al.(1987) and the recent reports from Bray et al. (2006).

The above experiments were performed using HEPES-buffered media. We repeated the studyusing a CO2/bicarbonated buffer system and atmospheric conditions of 5% CO2 and 5% O2,used for in vitro culture of the parasite (Trager and Jensen, 1976) and resembling the partialgas pressures present in venular capillaries where parasitized erythrocytes adhere. Again, pre-loaded chloroquine was rapidly released from Dd2, but slowly from HB3, with rates of 0.54 ±0.05 min−1 and 0.09 ± 0.03 min−1 respectively (Fig. S1). As the phenomena and rates wereindependent of the buffering system of the media in which experiments were performed, weconducted all subsequent kinetic studies using HEPES-buffered media.

The addition of 10 μM verapamil to the assay at t0 (the time at which efflux commenced, i.e.the cells were transferred to the external medium) eliminated the difference in chloroquineefflux rates between the two parasites (Fig. 1B), consistent with previous reports (Krogstad etal., 1987;Bray et al., 2006) (Fig. S2 further explores this effect by examining differentverapamil concentrations). In control experiments, we investigated whether verapamil exertsa cytotoxic effect during the 10 min time-course of the efflux studies. Erythrocytes infectedwith trophozoites of HB3 and Dd2 were exposed to verapamil concentrations ranging from 0to 100 μM for 10 min, washed and then returned to culture for 48 h (with [3H]-hypoxanthineadded for the full duration). Verapamil-treated and untreated parasites showed no differencein growth (Fig. S3), suggesting that P. falciparum can tolerate short-term exposure to highconcentrations of verapamil.

When the efflux experiments were repeated using glucose-deprived cells (replacing glucoseby an equal amount of 2-deoxyglucose), the differences in efflux kinetics between HB3 andDd2 vanished (Fig. 1C), consistent with previous reports (Bray et al., 2006). Chloroquine wasrapidly released from both HB3 and Dd2, with rates of 0.28 ± 0.02 min−1 and 0.23 ± 0.03min−1 respectively (Table 1). Addition of verapamil (10 μM) to the assay at t0 had no apparenteffect on the chloroquine efflux kinetics from glucose-deprived cells of either parasite(compare Fig. 1C and D and Table 1). The efflux experiments were performed under conditionswhere both HB3 and Dd2 parasites accumulated comparable levels of pre-loaded labelledchloroquine at t0. This involved pre-incubation in medium containing 40 nM [3H]-chloroquinefor both parasites.

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Previous studies have suggested that parasites deprived of glucose can no longer maintain theirintracellular ATP pool (Sherman, 1998), resulting in a breakdown of intracellular ion gradients,including the pH gradient across the food vacuolar membrane (Saliba and Kirk, 1999; Salibaet al., 2003). Under the glucose-deprived conditions employed in this study (10 min ofincubation in glucose-free medium), parasites are indeed unable to maintain an acidified foodvacuole, as demonstrated by their inability to accumulate the acidotropic fluorochromeLysoSensor Blue DND-192 (LS Blue) in this organelle (Fig. 2A and B) (Wissing et al.,2002; Rohrbach et al., 2005; Sanchez et al., 2005). Furthermore, ATP levels were substantiallyreduced in both HB3 and Dd2 parasites (Fig. 2C). Addition of glucose (after 10 min ofincubation in glucose-free medium) established both intracellular ATP levels and food vacuolarLS Blue accumulation within 6 min (6 min data points in Fig. 2), indicating that the cells remainviable within the 10 min time frame of the efflux experiments, despite lacking metabolicenergy.

Chloroquine efflux under ‘reverse varying-trans’ conditionsWe next investigated the kinetics of chloroquine efflux using the established ‘reverse varying-trans’ protocol (Levine et al., 1965; Baker and Widdas, 1973; Ribbes et al., 1985; Jarvis,1986; Stein, 1986; Byers et al., 1990; Zhou et al., 1992; Closs et al., 1993; Villalobos andBraun, 1998; Yabuuchi et al., 1999; Engel and Wang, 2005). To this end, cells incubated inglucose-rich media were preloaded with labelled chloroquine (see above) and then placed inmedium containing 0, 0.025 μM (HB3 only), 0.1 μM or 1.0 μM cold chloroquine. The presenceof extracellular chloroquine had a clear and concentration-dependent effect on the rate oflabelled chloroquine efflux in both HB3 and Dd2 parasites (compare circles – no externalchloroquine – with squares and triangles – with external chloroquine added, in each figure)bringing about a substantial increase in the release of label (Fig. 3A and B).

The addition of 10 μM verapamil to the assay at time t0 increased the amount of labelledchloroquine that was released in the presence of 0.1 μM trans-chloroquine in HB3 (compareclosed with grey squares in Fig. 3A). In contrast, in the CQR parasite Dd2, verapamilsignificantly inhibited stimulated chloroquine efflux in the presence of 0.1 μM extracellularchloroquine (1.20 ± 0.06 min−1 versus 0.50 ± 0.04 min−1; compare open with grey squares inFig. 3B).

Stimulation of labelled chloroquine efflux by extracellular chloroquine was also observed inthe absence of metabolic energy. The presence of 0.1 μM and 1.0 μM cold chloroquine in theexternal medium (compare circles – no external chloroquine – with squares and triangles –withexternal chloroquine added, in each figure) stimulated pre-loaded chloroquine efflux from bothHB3 and Dd2 parasites (Fig. 4A and B). Interestingly, the rates of labelled chloroquine effluxand the effects of added external chloroquine were comparable in both parasites under theseconditions (Table 2 and Fig. 4). Stimulated chloroquine efflux was insensitive to 10 μMverapamil in glucose-deprived cells (compare open with grey squares in Fig. 4B).

Association of chloroquine efflux kinetics with the PfCRT K76T mutationTo assess the contribution of PfCRT polymorphisms to the kinetics of chloroquine efflux, weinvestigated the previously described parasite clone N3D6 (Lakshmanan et al., 2005). N3D6is isogenic to Dd2, with the exception that the N3D6 harbours a PfCRT T76K substitution,which was introduced by allelic exchange, leading to the restoration of the wild-type aminoacid lysine. Unlike Dd2, N3D6 is susceptible to chloroquine (chloroquine IC50 = 17.0 ± 2.0nM). In the presence of glucose, preloaded labelled chloroquine was slowly released fromN3D6, with a rate of 0.04 ± 0.01 min−1, comparable to HB3 parasites assayed under similarconditions (Fig. 5A closed circles; Table 1; see Fig. 1A for comparison). The addition of 10μM verapamil slightly increased chloroquine release from N3D6 (compare closed with grey

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circles in Fig. 5A). In the absence of glucose, chloroquine efflux was rapid and insensitive to10 μM verapamil with a rate of 0.34 ± 0.07 min−1 (Fig. 5B, compare open with grey circles;Table 1). Extracellular chloroquine (at 0.1 μM, squares, and at 1.0 μM, triangles in Fig. 5Aand B) substantially stimulated chloroquine efflux, both in the presence and absence of glucose,again similar to HB3 (Fig. 5A and B; Table 2).

Trans-chloroquine increased apparent initial rates of chloroquine effluxTo better assess the effect of external chloroquine on chloroquine efflux, we plotted theapparent initial efflux rates as a function of the external chloroquine concentration (Fig. 6). Inall cases, a hyperbolic relationship was found, with the efflux rates significantly increasingwith each increment of external cold chloroquine added until a plateau was reached, as wouldbe expected for a carrier-mediated process (Levine et al., 1965;Baker and Widdas, 1973;Ribbeset al., 1985;Jarvis, 1986;Stein, 1986;Byers et al., 1990;Zhou et al., 1992;Closs et al., 1993).In the absence of glucose (Fig. 6A), the curves are comparable for HB3 (closed circles), Dd2(open circles) and N3D6 (closed inverted triangles), suggesting a common mechanism. Onlyin the presence of metabolic energy do the curves differ, with the CQR parasite Dd2consistently displaying higher apparent initial efflux rates as compared with the CQS parasitesHB3 and N3D6 (Fig. 6B).

DiscussionThe key finding of our study is that extracellular chloroquine can stimulate the efflux of pre-loaded chloroquine from both CQS and CQR parasites in the presence and absence of metabolicenergy. As parasites deprived of glucose can no longer maintain their intracellular ATP pool(Fig. 2C) (Sherman, 1998) or the pH gradient across the food vacuolar membrane (Fig. 2)(Saliba and Kirk, 1999;Saliba et al., 2003), the stimulation effect cannot be explained byincoming cold chloroquine titrating the intra-food vacuolar protons, which in turn would leadto rapid release of labelled pre-loaded chloroquine. Could stimulated chloroquine efflux resultfrom a displacement effect? In vitro experiments have shown that the addition of coldchloroquine can release labelled chloroquine from parasite-derived cell debris containinghaem, hemozoin and other components, such as nucleic acids, membranes and proteins, towhich chloroquine can bind at the pH of 7.4 used in the cited study (Bray et al., 1998). Invivo, however, both CQS and CQR parasites have a high chloroquine binding capacity ofapproximately 30 μmol per litre parasite volume (Bray et al., 1998;Sanchez et al., 2003), whichdoes not seem to be saturated under the experimental conditions used in this study. Pre-equilibrating parasites with 100 nM of cold chloroquine (a trans-chloroquine concentrationrevealing stimulated efflux, Figs 3–5) followed, after washing, by an incubation with 40 nMof labelled chloroquine (a concentration used for pre-loading in the efflux studies) showed nosignificant differences in the amount of label taken up by CQS parasites as compared withcontrol cells that had not been pre-equilibrated with unlabelled chloroquine (Sanchez et al.,2003;2005). In CQR parasites, the accumulation of labelled chloroquine was even increasedunder these conditions (Sanchez et al., 2003;2005) (see below for further discussion). On thebasis of these results, we do not favour the hypothesis that stimulated efflux is due to incomingcold chloroquine displacing labelled chloroquine from haem binding sites.

The observed increase in the apparent initial efflux rates, with its hyperbolic dependence onthe concentration of external chloroquine (Fig. 6), is instead indicative of trans-stimulation(Baker and Widdas, 1973;Ribbes et al., 1985;Jarvis, 1986;Stein, 1986;Byers et al., 1990;Zhouet al., 1992;Closs et al., 1993;Villalobos and Braun, 1998;Yabuuchi et al., 1999;Engel andWang, 2005). Trans-stimulation is an intrinsic characteristic of a carrier and is generallyexplained by substrates in trans re-orienting the carrier such that the transport cycle and thecis- to trans-movement of substrate is accelerated (Stein, 1986). On the basis of these data, we

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propose that both CQS and CQR parasites possess a carrier of chloroquine, but with differenttransport properties, as will be discussed below.

Chloroquine is a diprotic, amphiphilic weak base that accumulates in the parasite’s foodvacuole, driven by acidotropic trapping (Yayon et al., 1984; 1985) and binding to haem (Chouet al., 1980; Bray et al., 1998). Considering a steady-state food vacuolar pH value ofapproximately 4.9–5.2 (Yayon et al., 1984; Krogstad et al., 1985; Hayward et al., 2006; Kuhnet al., 2007), chloroquine would accumulate up to 20 000-fold, assuming simple passivediffusion of the free base and a membrane impermeable to both mono- and di-protonatedchloroquine. In CQS parasites, chloroquine can indeed accumulate to such levels [calculatingusing a volume of 4 fl and 75 fl for the food vacuole (Sullivan et al., 1996) and the trophozoite-infected erythrocyte (Saliba et al., 1998) respectively, and a cellular accumulation ratio of1200-fold (Bray et al., 1998; 2006; Sanchez et al., 2003)].

Applying the simple partitioning model, an increased food vacuolar pH value, which once wasconsidered a model of chloroquine resistance (Yayon et al., 1984), could explain the rapidchloroquine efflux associated with CQR parasites (under zero-trans conditions and in thepresence of metabolic energy). As less chloroquine is protonated at a higher pH, morechloroquine base is available and will flow out of the food vacuole and the cell by passivediffusion across cellular membranes. However, two independent studies have recentlyquantified the steady-state pH of the food vacuole and found no significant differences betweenCQS and CQR parasites (Hayward et al., 2006; Kuhn et al., 2007). Other studies have reportedfood vacuolar pH values that, paradoxically, are lower in CQR than in CQS parasites(Dzekunov et al., 2000; Bennett et al., 2004). These data are difficult to reconcile with therecent food vacuolar pH measurements (Hayward et al., 2006; Kuhn et al., 2007) and,furthermore, are inconsistent with our efflux studies. According to the simple partitioningmodel, at a lower pH, more chloroquine is present in its protonated forms and less as free base.This would result in a reduced chloroquine efflux, as compared with CQS parasites, which iscontrary to the actual findings (Fig. 1). Thus, altered food vacuolar pH cannot explain the rapidchloroquine efflux observed in CQR parasites.

Alternatively, differences in haem binding kinetics brought about by altered haem bio-mineralization and/or turn over rates (Dzekunov et al., 2000; Ursos et al., 2001; Ursos andRoepe, 2002) or the acquisition of a haem binding protein (Bray et al., 1998) could accountfor the rapid chloroquine efflux in CQR parasites. If differences in haem binding kineticsexisted, one might expect CQR parasites to take up less chloroquine, irrespective of thepresence of metabolic energy or a standing trans-food vacuolar pH gradient. However, this iscontrary to the experimental findings. In this and other studies, it has been shown that CQRand CQS parasites accumulate comparable amounts of chloroquine in the absence of glucose(Fig. 3) (Krogstad et al., 1992; Sanchez et al., 2003; 2004; Bray et al., 2006) or in the presenceof the ionophore FCCP (Bray et al., 2006), which dissipates proton gradients.

Additional evidence argues against altered haem binding or haem bio-mineralization kineticsbeing responsible for altered chloroquine accumulation and efflux. Naude et al. (2005) haverecently transferred the key feature of chloroquine resistance, namely, reduced verapamil-sensitive chloroquine accumulation, to a haem-and hemozoin-free system, by expressingPfCRT in acidic vesicles of the amoeba Dictyostelium discoideum. Vesicles expressing theCQR PfCRT variant accumulated less chloroquine in a verapamil-sensitive manner thanuntransfected controls or vesicles containing wild-type PfCRT (Naude et al., 2005).

If the differences in chloroquine accumulation and efflux observed between CQS and CQRparasites do not arise from altered haem binding or drug partitioning, an alternative mechanismmust be present. Proposed models include a carrier (Krogstad et al., 1987; Fidock et al.,

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2000; Sanchez et al., 2003; 2004; 2005; Naude et al., 2005) or a channel of chloroquine(Warhurst et al., 2002; Zhang et al., 2004; Bray et al., 2006).

In previous studies, we investigated the effect of trans-chloroquine on chloroquineaccumulation and found that low concentrations of pre-loaded cold chloroquine was able tostimulate the chloroquine uptake maximum in CQR parasites (Sanchez et al., 2003; 2004;2005). Consistent with established theorems in membrane transport, we explained thisobservation by the presence of a saturable carrier of chloroquine present in these parasites. Iflabelled chloroquine is being transported out of the food vacuole in the absence of pre-loadedchloroquine, this outward transport can be competed for by unlabelled chloroquine, present atthe membrane face from which label is being transported. This evidence in favour of a carrierof chloroquine is corroborated by the finding of trans-stimulated efflux, in which extracellularchloroquine increased the apparent initial rate of chloroquine efflux in CQR parasites (Fig. 6).Trans-stimulated chloroquine efflux responded to verapamil in CQR parasites (Fig. 3B),indicating that the phenomenon is specific and not brought about by an increase in foodvacuolar pH due to incoming chloroquine. Thus, transport experiments performed in the twovectorial directions (accumulation and efflux conditions) provide strong evidence for thepresence of a carrier for chloroquine in CQR parasites.

Unexpectedly, CQS parasites also showed evidence of a carrier for chloroquine. Like in CQRparasites, extracellular chloroquine stimulated the apparent initial rates of pre-loadedchloroquine efflux in both the presence and absence of metabolic energy (Fig. 6), consistentwith trans-stimulation of a carrier. Such trans-stimulation was clearly evident in one vectorialdirection, under efflux (Fig. 6), and less obvious in accumulation conditions (Sanchez et al.,2003;2005). The ‘varying-trans’ (accumulation) and the ‘reverse varying-trans’ (efflux)procedure producing different outcomes is not unusual, and has been demonstrated in manyother carrier systems (Sen and Widdas, 1962;Hoare, 1972;Cabantchik and Ginsburg,1977;Stein, 1986). It can be explained by different affinities of the carrier for the solute at theinner and outer face of the membrane (Sen and Widdas, 1962;Hoare, 1972;Cabantchik andGinsburg, 1977;Stein, 1986). Circumstantial evidence indeed point towards a low-affinitycarrier of chloroquine present in CQS parasites, as suggested by a comparative analysis of thetime-courses of cellular chloroquine accumulation in the presence of preloaded chloroquine(Sanchez et al., 2003). For illustrative purposes, these time-courses are reproduced fromSanchez et al. (2003) (Fig. S4A). The time-courses show a rise and fall in both CQS and CQRparasites as the experiment proceeded, with the peaks of labelled chloroquine uptake greatlyexceeding the steady-state level of chloroquine accumulation (Fig. S4A). This phenomenon isreminiscent of counter-transport, as originally defined by GLUT1-mediated glucose transportin human erythrocytes (Fig. S4B) (Baker and Widdas, 1973). As the time-courses are onlycomparable when substantially more chloroquine is pre-loaded in CQS parasites (1.22 ± 0.16mM versus 0.021 ± 0.001 mM in CQR parasites), this would suggest that the carrier in CQSparasites has a lower affinity for chloroquine than CQR parasites.

The rapid chloroquine efflux revealed by CQR parasites under zero-trans conditions and inthe presence of metabolic energy was causatively linked to the K76T polymorphism withinPfCRT (Fig. 5). The Dd2 back-mutant N3D6 did not show this rapid efflux. N3D6 differs fromDd2 only in a single amino acid substitution within PfCRT, replacing the mutant threonine atposition 76 with the wild-type lysine (Lakshmanan et al., 2005). This identifies PfCRT as acandidate for the chloroquine carrier.

Contrasting with the carrier model, Warhurst et al. (2002) and Bray et al. (2006) have proposedthat PfCRT acts as a channel of chloroquine, allowing protonated chloroquine to passivelyflow down its concentration gradient and out of the food vacuole (Warhurst et al., 2002; Zhanget al., 2004; Bray et al., 2006). According to this model, the K76T mutation within PfCRT,

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which replaces a positively charged lysine with an uncharged threonine within thetransmembrane domain 1, removes an electrostatic charge that prohibits protonated and hence,positively charged chloroquine from entering the channel (Warhurst et al., 2002; Zhang etal., 2004; Bray et al., 2006). Only when the positive charge is removed is the channel accessibleto charged chloroquine (Warhurst et al., 2002; Zhang et al., 2004; Bray et al., 2006). Thismodel was evoked to explain why, in the absence of metabolic energy or in the presence of theproton uncoupler FCCP, chloroquine efflux is equally rapid in both CQS and CQR parasites(Bray et al., 2006), as also shown in the present study (Fig. 4; Table 1). But an alternativeexplanation is possible: as glucose deprivation and the addition of FCCP both break down thefood vacuolar pH gradient (Saliba and Kirk, 1999; Saliba et al., 2003), which is responsiblefor acidotropic trapping of chloroquine (Yayon et al., 1984; 1985), a far larger fraction ofchloroquine will be unprotonated at the now neutral pH value. Accordingly, more chloroquinebase is able to rapidly flow out of the cell by simple diffusion in both CQS and CQR parasites.Thus, the behaviour of chloroquine movement in the absence of metabolic energy or thepresence of FCCP under zero-trans conditions does not differentiate between a carrier and achannel.

A distinguishing feature is the trans-stimulation kinetics that we have presented in this andprevious studies (Sanchez et al., 2003; 2004; 2005). Facilitated diffusion through aconventional pore or channel does not demonstrate trans-stimulation, because movements indifferent directions within a pore or channel block rather than enhance one another (Keynesand Lewis, 1950; Dani and Levitt, 1981; Stein, 1986). On the other hand, trans-stimulation isindicative of a carrier-mediated process (Baker and Widdas, 1973; Ribbes et al., 1985; Jarvis,1986; Stein, 1986; Byers et al., 1990; Zhou et al., 1992; Closs et al., 1993). As both CQS andCQR parasites revealed trans-stimulated chloroquine efflux, we propose that both parasitespossess a carrier of chloroquine. In metabolically starved cells, the apparent initial efflux ratesare comparable in both parasites (Fig. 6A), suggesting that the putative carriers have similarproperties in the absence of metabolic energy. The carriers are insensitive to verapamil andresemble a simple reversible carrier. Because of distinct kinetic properties, it is unlikely thatthe proposed chloroquine carrier identifies with the erythrocyte plasma membrane chloroquinecarrier described by Yayon and Ginsburg (1982). For example, the zero-trans apparent initialefflux rates differ approximately 20-fold [0.28 min−1 at 37°C (this study) versus 5.19 min−1

at 25°C (Yayon and Ginsburg, 1982)]. We therefore reason that the trans-stimulatedchloroquine efflux observed in both CQS and CQR parasites, under glucose deprivation, relatesto a carrier of parasite origin.

In the presence of metabolic energy the kinetic properties of the parasite-derived chloroquinecarriers fundamentally differ between CQS and CQR parasites, as demonstrated by thedifferences in the apparent initial efflux rates (Fig. 6B). CQR parasites consistently revealedhigher apparent initial efflux rates (Fig. 6B), suggesting a more efficient efflux carrier ofchloroquine. Moreover, in CQR parasites, the carrier responds to verapamil in the presence ofmetabolic energy (Figs 1B and 3B), while the carrier in CQR parasites does not (Figs 1B and3A).

Our model of a chloroquine carrier is consistent with PfCRT being a member of the drug/metabolite transporter superfamily (Martin and Kirk, 2004; Tran and Saier, 2004), whichcomprises secondary active co- and counter-transporters. The postulated carrier may transporteither protonated chloroquine from the food vacuole’s interior or chloroquine base from thefood vacuolar membrane (preemptive transport). The K76T transition in PfCRT may increasethe affinity of the carrier for chloroquine (Fig. S4), or, alternatively, couple outwardchloroquine transport to the proton-motive force. Verapamil may reverse these effects bysubstituting for the positively charged lysine when bound to PfCRT. Single amino acid changescan affect the linkage of a transport process to a secondary energy source, as demonstrated by

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the rabbit proton-peptide co-transporter PepT1 (Meredith, 2004) and the Escherichia coliNa+/proline co-transporter (Quick et al., 1999). Linking chloroquine efflux to the proton-motive force, generated by the trans-food vacuolar proton gradient, would explain therequirement of an energy input to reduce chloroquine accumulation in CQR parasites belowlevels observed in the absence of metabolic energy (Krogstad et al., 1992; Sanchez et al.,2003; 2004; Bray et al., 2006). This also explains the effect of the ionophore FCCP, whichincreases the levels of chloroquine accumulation in CQR parasites (Bray et al., 2006),suggesting that a standing pH gradient is required to drive out chloroquine. It is the trappingof charged chloroquine in the acidic food vacuole that we assert is overcome by the active,PfCRT-mediated transport process. Note, in the absence of metabolic energy there is noacidotropic trapping of chloroquine. The equilibrium of the different chloroquine forms isshifted from the membrane impermeable protonated forms to the now membrane permeablefree base, which rapidly flows out of the cell. Thus, when comparing efflux rates, the pH ofthe food vacuole and its effect on the distribution of protonated versus unprotonatedchloroquine need to be considered. The twofold increase in the apparent initial efflux rate from0.23 ± 0.03 min−1 to 0.55 ± 0.06 min−1 in the absence and presence of metabolic energy inDd2 parasites (Table 2) is consistent with our model. A carrier would require specificinteractions with the solute it transports. The recently reported binding of PfCRT to chloroquine(Zhang et al., 2004) and the finding that selection to different antimalarial drugs causes sets ofdifferent point mutations within PfCRT (Fidock et al., 2000; Cooper et al., 2002; 2006; Sidhuet al., 2002; Ferdig et al., 2004; Johnson et al., 2004; Lakshmanan et al., 2005) fully accordwith this view and our carrier model. In summary, our data provide strong evidence in favourof a PfCRT-mediated carrier of chloroquine being the basis of chloroquine resistance in P.falciparum.

Experimental proceduresMaterial

[3H]-chloroquine (18.8 Ci mmol−1), [3H]-hypoxanthine (17.9 Ci mmol−1) and NCS-II tissuesolubilizer were supplied by Amersham Biosciences. Blasticidin was purchased fromInvioGen. Chloroquine, 2-deoxyglucose and verapamil were from Sigma. Media werepurchased from Invitrogen. The following media were used: (i) complete RPMI 1640; (ii)bicarbonate-free RPMI 1640; and (iii) glucose- and bicarbonate-free RPMI 1640. All mediawere supplemented with 25 mM HEPES-Na and 2 mM glutamine.

Parasite culture and characterizationPlasmodium falciparum parasites were cultured as described (Trager and Jensen, 1976) andsynchronized using the sorbitol method (Lambros and Vanderberg, 1979). The parasite cloneN3D6 was grown in medium supplemented with 2.5 μg ml−1 blasticidin (Lakshmanan et al.,2005). One day preceding the experiment, the drug was removed from the culture media. Thegenotype of all parasite clones was confirmed using microsatellite analysis of the PfRRMmarker (Su et al., 1998).

Reverse varying-trans procedurePlasmodium falciparum-infected erythrocytes (at the trophozoite stage) were purified using astrong magnet, as described (Sanchez et al., 2003). This yielded 98–100% purified trophozoite-infected erythrocytes, as directly determined by microscopic examination of Giemsa-stainedblood smears. Lysis of infected erythrocytes was not observed. Purified infected erythrocyteswere resuspended in the appropriate medium at a haematocrit of approximately 25 000 cellsμl−1. The exact haematocrit was determined using a Neubauer counting chamber. The ‘reversevarying-trans’ efflux procedure consists of a pre-loading phase during which the P.falciparum parasites HB3, N3D6 and Dd2 were incubated at 37°C for 15 min until equilibrium

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(Sanchez et al., 2003) in medium containing the appropriate amount of [3H]-chloroquine. The[3H]-chloroquine concentration in the pre-incubation medium varied depending on the parasiteused and the condition applied, and had been empirically determined to ensure comparableintracellular drug concentrations at the end of the pre-loading phase (data not shown). Inglucose-rich medium, HB3, N3D6 and Dd2 were incubated in medium containing 20 nM (bothHB3 and N3D6) and 100 nM (Dd2) chloroquine respectively. Under glucose-deprivedconditions, HB3, N3D6 and Dd2 were incubated in glucose-free medium containing 40 nM of[3H]-chloroquine. After pre-loading, cells were washed twice in ice-cold medium (pH adjustedto 7.3 at 4°C) and resuspended in pre-warmed medium containing 0, 0.025 μM, 0.1 μM or 1.0μM unlabelled chloroquine. The reaction was held at 37°C and the time-course of [3H]-chloroquine efflux was monitored. In cases where bicarbonated RPMI 1640 medium was used,the entire experiment including pre-loading with labelled chloroquine was conducted undercontrolled atmospheric conditions of 5% CO2 and 5% O2 using a gassed incubation chamber.Duplicate 150 μl aliquots were removed from the reaction at various time points andimmediately spun through a layer of a 5:4 mixture of dibutyl phthalate and dioctyl phthalate(15 000 g, 5 s) to separate the cells from the aqueous medium that contained the extracellular[3H]-chloroquine. The cell pellets were recovered by cutting the reaction tubes through the oillayer. The amount of extracellular and intracellular [3H]-chloroquine was determined using aliquid scintillation counter (TRI-CARB 2100 TR, Packard), as described (Sanchez et al.,2003). The intracellular [3H]-chloroquine concentration was calculated from the amount ofintracellular label by assuming that the volume of a trophozoite-infected erythrocyte is 75 fl(Saliba et al., 1998), and normalizing the measurements to 1 × 106 infected erythrocytes. Whereindicated, verapamil was added to the reaction at the indicated concentration.

Curve fittingKinetic data were analysed using a computerized least squares fit method (Sigma Plot 9, JandelCorporation). For the release kinetics study we used the exponential decay algorithm fromSigma Plot, transformed to calculate the apparent initial rates of release directly, this beingequal to a*b in the SigmaPlot exponential curve fit equation. The equation used is:CQremaining = CQ∞ + (Init/b)*exp(−bt), where t is the time during which efflux is occurring,b is the rate constant for efflux and Init is the apparent initial rate of efflux. When t = 0, theCQremaining has the value CQ∞ + Init/b, while the apparent initial rate is given by Init, whichis the value of the slope as t tends to zero. At infinite time, CQremaining has the value CQ∞.Rates were derived for the amount of exchangeable label, as defined by extrapolating the effluxcurves to infinite time, with the total amount of chloroquine efflux computed using the initialisotopic specific activity.

Statistical testsTests of significant differences between derived parameters were performed using Student’st-test, with significance set at P < 0.05.

Measurement of intracellular ATPThe concentration of ATP within the parasite was measured using the ATP-Assay kit(Calbiochem), as described by the manufacturer. Briefly, 1 × 106 magnet purified infectederythrocytes (at the trophozoite stage) were resuspended in 90 μl of the nucleotide releasingbuffer provided by the kit. Ten microlitres of whole cell lysate was diluted in 290 μl of HEPESbuffer solution. One hundred microlitres of luciferase reagent was added and the luciferaseactivity determined using a luminometer (Lumat LB 9507, EG & G Berthold, Germany).

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Live cell imagingLive cell imaging with the acidotropic dye LS Blue (Molecular Probes) was performed aspreviously described (Wissing et al., 2002; Rohrbach et al., 2005; Sanchez et al., 2005).Briefly, P. falciparum-infected erythrocytes were washed twice with Ringer solution (122.5mM NaCl, 5.4 mM KCl, 1.2 mM CaCl2, 0.8 mM MgCl2, 11 mM D-glucose, 10 mM HEPES,1 mM NaH2PO4, pH 7.4) and allowed to settle onto poly-L-lysine-coated coverslips in amicroperfusion chamber (Wunsch et al., 1998). Cells were then perfused with Ringer solutioncontaining 1 μM LS Blue. Confocal scanning fluorescence microscopy was performed usinga Zeiss LSM510 (Carl Zeiss, Germany) microscope equipped with UV laser lines (351 nm,364 nm) and an Axiovert 100M microscope. A pinhole of 1 μm was chosen to ensure thatfluorescence was from within the food vacuole and not from the regions lying above or below.LS Blue was excited at 364 nm and the emission was detected using an LP 385 nm filter. Imageacquisition was optimized to minimize the bleaching of the non-ratiometric dye LS Blue. Forquantification of the measurements, a region of interest (ROI) was chosen lying within the foodvacuole. The mean fluorescence of LS Blue was determined within the ROIs and plotted.

Short-term tolerance to verapamilHighly synchronized P. falciparum-infected erythrocytes at the trophozoite stage (parasitaemiaof 2–3% and a haematocrit of 5%) were washed using RPMI 1640 medium, resuspended inthe same medium and then incubated for 15 min at 37°C with verapamil concentrations rangingfrom 0 to 100 μM. After incubation, cells were washed twice, returned to culture and aproliferation assay was performed by incubating the cells for 48 h (an entire cell cycle includingreinvasion, from the trophozoite stage to the trophozoite stage) in the presence of [3H]-hypoxanthine (Desjardins et al., 1979).

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsThis work was supported by the Landesstiftung Baden-Württemberg, the SFB 544 entitled ‘Control of tropicalinfectious diseases’, and the European Commission (M.L.) and the NIH (AI50234, D.A.F.). We thank Elisabeth Wilkenand Marina Müller for technical assistance. This paper is dedicated to the memory of William Robert Lieb, a pioneerin the study of membrane transport kinetics and a dear friend, who died in tragic circumstances on 25 January, 2005.

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Fig. 1.Time-courses of chloroquine efflux from P. falciparum-infected erythrocytes pre-loaded with[3H]-chloroquine. A and B. In the presence of glucose: HB3 (closed circles), Dd2 (open circles)with (A) in the absence and (B) in the presence of 10 μM verapamil added at time t0 to theassay. C and D. Using glucose-deprived cells: (C) HB3 (closed triangles); Dd2 (open triangles);no verapamil added. (D) HB3 (closed triangles); Dd2 (open triangles); with 10 μM verapamiladded at time t0 to the assay. The means ± SEM of at least three independent determinationsare shown. The data points in this and the following figures were fitted using a simpleexponential decline and the rate constants compiled in Table 1.

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Fig. 2.Effect of glucose deprivation on LS Blue fluorescence and intracellular ATP. A. Erythrocytesinfected with the CQS parasite HB3 were perfused with glucose-free medium containing thequalitative pH indicator LS Blue. At t0, glucose was added to the medium (final concentration11 mM). Confocal images taken just prior to and 6 min after the addition of glucose are shown.LS Blue accumulates in the food vacuole resulting in a bright fluorescence arising from thisorganelle (here shown in blue). Images are also available for Dd2, but are not shown in thisfigure. Bar, 5 μm.B. Quantification of food vacuolar LS Blue fluorescence (FLS Blue), provided as arbitrary units(a.u.), before and 6 min after the addition of glucose to the medium. Control indicates foodvacuolar LS Blue fluorescence intensities determined for cells held in glucose-supplementedmedium during the entire duration of the experiments. Left panel, HB3; right panel, Dd2. Themeans ± SEM are shown for 30 or more determinations.

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C. The intracellular ATP concentration was measured before and 6 min after the addition ofglucose to the medium. The ATP concentration of control cells held in glucose-rich mediumduring the entire duration of the experiment is shown for comparison. Left panel, HB3; rightpanel, Dd2. The means ± SEM are shown for at least four independent determinations.

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Fig. 3.Chloroquine efflux kinetics under ‘reverse varying-trans’ conditions, in glucose-rich medium.P. falciparum-infected erythrocytes were pre-loaded with comparable amounts of [3H]-chloroquine, washed and placed in medium containing the following concentrations of coldchloroquine: 0.0 μM (circles), 0.025 μM (inverted triangles), 0.1 μM (squares), 1.0 μM(triangles).A. CQS parasite HB3.B. CQR parasite Dd2.In both panels, the grey squares (0.1 μM external chloroquine) show the effect of addition of10 μM verapamil at zero time (dotted line). The means ± SEM of at least three independentdeterminations are shown.

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Fig. 4.Chloroquine efflux kinetics under ‘reverse varying-trans’ conditions, in glucose-free medium.P. falciparum-infected erythrocytes were pre-loaded with comparable amounts of [3H]-chloroquine, washed and placed in glucose-free medium (replacing glucose by an equal amountof 2-deoxyglucose) containing the following concentrations of cold chloroquine: 0.0 μM(circles), 0.1 μM (squares), 1.0 μM (triangles).A. CQS parasite HB3.B. CQR parasite Dd2.The grey squares (0.1 μM external chloroquine) show the effect of addition of 10 μM verapamilat zero time (dotted line). The means ± SEM of at least three independent determinations areshown.

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Fig. 5.Time-courses of chloroquine efflux from the CQS parasite N3D6 under ‘reverse varying-trans’ conditions. Chloroquine efflux experiments were performed under (A) glucose-rich or(B) glucose-deprived conditions. Cells were loaded with labelled chloroquine and placed inthe appropriate medium containing the following concentrations of cold chloroquine: 0.0 μM(circles), 0.1 μM (squares), 1.0 μM (triangles). The zero-trans control was repeated in thepresence of 10 μM verapamil (grey circles, dotted line). The means ± SEM of three independentdeterminations are shown.

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Fig. 6.Apparent initial rates of chloroquine efflux as a function of the external cold chloroquineconcentration under ‘reverse varying-trans’ conditions. Apparent initial rates were directlycomputed from the relevant efflux curves of Figs 3–5, as described in Experimentalprocedures. CQS parasite HB3, closed circles; CQR parasite Dd2, open circles; CQS parasiteN3D6, inverted closed triangles.A. Glucose-deprived cells.B. Glucose-supplemented cells.The lines drawn are best-fits to the three-parameter hyperbolic algorithm using the SigmaPlotsoftware.

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Table 1

Apparent initial chloroquine efflux rates from various P. falciparum parasites in glucose-rich (Glu) or glucose-free medium (replaced by an equivalent amount of 2-deoxyglucose, 2DG), and in the presence and absence of10 μM verapamil (Ver).

Parasite

Glu 2DG

−Ver (min−1) +Ver (min−1) −Ver (min−1) +Ver (min−1)

Dd2 (CQR) 0.55 ± 0.06* (5) 0.08 ± 0.01 (4) 0.23 ± 0.03** (3) 0.25 ± 0.03** (3)

HB3 (CQS) 0.09 ± 0.04 (9) 0.08 ± 0.01 (3) 0.28 ± 0.02** (3) 0.31 ± 0.03** (3)

N3D6 (CQS) 0.04 ± 0.01 (3) 0.07 ± 0.01 (3) 0.34 ± 0.07** (3) 0.36 ± 0.09** (3)

The means ± SEM of n independent determinations are shown.

*P < 0.01, with HB3 as a reference;

**P < 0.01 between rates determined in the presence and absence of glucose.

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Tabl

e 2

App

aren

t ini

tial c

hlor

oqui

ne e

fflu

x ra

tes f

rom

var

ious

P. f

alci

paru

m p

aras

ites u

nder

‘rev

erse

var

ying

-tran

s’ c

ondi

tions

in g

luco

se-r

ich

(Glu

) or g

luco

se-f

ree

med

ium

(rep

lace

d by

an

equi

vale

nt a

mou

nt o

f 2-d

eoxy

gluc

ose,

2D

G).

Tran

s-C

Q (μ

M)

HB

3D

d2N

3D6

Glu

(min−1

)2D

G (m

in−1

)G

lu (m

in−1

)2D

G (m

in−1

)G

lu (m

in−1

)2D

G (m

in−1

)

00.

09 ±

0.0

40.

28 ±

0.0

20.

55 ±

0.0

60.

23 ±

0.0

30.

05 ±

0.0

10.

34 ±

0.0

8

0.02

50.

10 ±

0.0

7n.

d.n.

d.n.

d.n.

d.n.

d.

0.1

0.47

± 0

.11

0.95

± 0

.08

1.20

± 0

.06

0.97

± 0

.05

0.30

± 0

.08

1.01

± 0

.12

1.0

1.00

± 0

.03

4.40

± 0

.45

2.99

± 0

.32

4.88

± 0

.48

1.14

± 0

.08

4.62

± 0

.58

Tran

s-ch

loro

quin

e (tr

ans-

CQ

) ref

ers t

o th

e co

ncen

tratio

n of

chl

oroq

uine

in th

e ou

tsid

e m

ediu

m. T

he m

eans

± S

EM o

f at l

east

thre

e in

depe

nden

t det

erm

inat

ions

are

show

n. n

.d.,

not d

eter

min

ed.

Mol Microbiol. Author manuscript; available in PMC 2010 September 23.