Chloroquine Transport in <i>Plasmodium falciparum</i>. 1. Influx and Efflux Kinetics for Live Trophozoite Parasites Using a Novel Fluorescent Chloroquine Probe

Cabrera, Mynthia; Natarajan, Jayakumar K.; Paguio, Michelle F.; Wolf, Christian; Urbach, Jeffrey S.; Roepe, Paul D.

doi:10.1021/bi901034r

Cited by 16 publications

(48 citation statements)

References 39 publications

(124 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As described [9], a CQ fluorescent analogue (NBD-CQ) was synthesized via reductive amination of N -t-Boc-glycinal with monodesethyl CQ. Drug susceptibility assays and confocal microscopy revealed that pharmacological activity and subcellular localization of the probe are similar relative to CQ for both CQS and CQR parasites.…”

Section: Resultsmentioning

confidence: 99%

“…Data in this paper and in the accompanying [9] provide the most direct evidence to date for PfCRT-mediated transport of quinoline compounds. NBD-CQ is a valid probe for CQ transport that behaves quite similar to CQ.…”

Section: Discussionmentioning

confidence: 99%

“…Kinetic resolution of the 3 H-CQ data is poor so we are unable to unequivocally conclude whether accumulation into Dd2 ISOV proceeds with any significantly different rate constant relative to control or HB3 ISOV. However, accumulation of NBD-CQ into live intact iRBC [9] clearly showed a reduced rate constant for CQR parasites. Similar to earlier interpretation [13], the ATP dependence for this effect could be (in part) reflecting a DV pH, ΔpH, or ΔΨ dependence for PfCRT-mediated outward transport since these parameters are controlled by cytosolic ATP.…”

Section: Discussionmentioning

confidence: 99%

“…These include that PfCRT mediates downhill diffusion of charged CQ [7] or CQ exchange [8], and that PfMDR1 mediates adenosine 5’-triphosphate (ATP) dependent transport at the digestive vacuole (DV) membrane [6]. In the accompanying paper [9], we show that the rate constant for DV accumulation of a fluorescent CQ analogue (NBD-CQ) is conspicuously reduced for CQR parasites, and that this is due to mutation of PfCRT protein to a CQR isoform. This suggests several possibilities for PfCRT function including: CQS isoform PfCRT performs ATP dependent CQ uptake (presumably secondary active transport since PfCRT does not directly hydrolyze ATP) and CQR PfCRT does not; or, CQR isoform PfCRT performs outward mediated drug transport at a rate that can compete with ATP dependent accumulation.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Chloroquine Transport inPlasmodium falciparum. 2. Analysis of PfCRT-Mediated Drug Transport Using Proteoliposomes and a Fluorescent Chloroquine Probe

Paguio

Cabrera²,

Roepe³

2009

Biochemistry

Self Cite

View full text Add to dashboard Cite

Mutations in the PfCRT protein cause chloroquine resistance (CQR) and earlier studies from our laboratory using plasma membrane inside-out vesicles (ISOV) prepared from yeast expressing recombinant PfCRT [Zhang, H., et al. (2004) Biochemistry 43, 8290–8296] suggested that the putative transporter mediates downhill facilitated diffusion of charged chloroquine (CQ). However, more recent experiments with a fluorescent CQ probe (NBD-CQ) presented in the accompanying paper [Cabrera, M., et al. (2009), XXXX-XXXX] indicated that the CQR phenotype in live parasites is associated with a reduced rate of ATP dependent CQ uptake into the digestive vacuole (DV). An altered rate constant for uptake has multiple interpretations. To further investigate this phenomenon, PfCRT proteins found in chloroquine sensitive (CQS) and CQR strains of Plasmodium falciparum were purified from yeast engineered to express “yeast optimized” pfcrt genes, reconstituted into proteoliposomes (PL), and efflux of NBD-CQ was measured from these PL. A membrane impermeant quencher was used to distinguish intra-PL NBD-CQ from extra-PL NBD-CQ vs. time as well as resolve initial rates and rate constants for efflux. Efflux was investigated at a range of NBD-CQ concentrations, in the presence vs. absence of pH gradients (ΔpH) and transmembrane potentials (ΔΨ). Explicit turnover numbers for apparent PfCRT-mediated transport were then calculated under these conditions. Our data are consistent with a model wherein PfCRT catalyzes electrochemically downhill diffusion of NBD-CQ out of the DV, in response to ΔΨ or ΔpH, at a rate that can partially compete with the ATP dependent uptake of NBD-CQ by CQS parasites described in the previous paper. These data allow us to propose a refined model for altered CQ accumulation in CQR malarial parasites.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Chloroquine Transport inPlasmodium falciparum. 2. Analysis of PfCRT-Mediated Drug Transport Using Proteoliposomes and a Fluorescent Chloroquine Probe

Paguio

Cabrera²,

Roepe³

2009

Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Mutant PfCRT has been postulated to act either as a channel, permitting the mediated and fast transport of diprotonated CQ across the DV membrane, or as an outwardly directed slower CQ carrier (6,26,35,39). The transport of GSH is time dependent, but appears to be nonsaturable, which could suggest that the transport is not carrier mediated.…”

Section: Discussionmentioning

confidence: 99%

Glutathione Transport: A New Role for PfCRT in Chloroquine Resistance

Patzewitz

Salcedo-Sora

Wong

et al. 2013

Antioxidants & Redox Signaling

View full text Add to dashboard Cite

Aims: Chloroquine (CQ) kills Plasmodium falciparum by binding heme, preventing its detoxification to hemozoin in the digestive vacuole (DV) of the parasite. CQ resistance (CQR) is associated with mutations in the DV membrane protein P. falciparum chloroquine resistance transporter (PfCRT), mediating the leakage of CQ from the DV. However, additional factors are thought to contribute to the resistance phenotype. This study tested the hypothesis that there is a link between glutathione (GSH) and CQR. Results: Using isogenic parasite lines carrying wild-type or mutant pfcrt, we reveal lower levels of GSH in the mutant lines and enhanced sensitivity to the GSH synthesis inhibitor l-buthionine sulfoximine, without any alteration in cytosolic de novo GSH synthesis. Incubation with N-acetylcysteine resulted in increased GSH levels in all parasites, but only reduced susceptibility to CQ in PfCRT mutant-expressing lines. In support of a heme destruction mechanism involving GSH in CQR parasites, we also found lower hemozoin levels and reduced CQ binding in the CQR PfCRT-mutant lines. We further demonstrate via expression in Xenopus laevis oocytes that the mutant alleles of Pfcrt in CQR parasites selectively transport GSH. Innovation: We propose a mechanism whereby mutant pfcrt allows enhanced transport of GSH into the parasite's DV. The elevated levels of GSH in the DV reduce the level of free heme available for CQ binding, which mediates the lower susceptibility to CQ in the PfCRT mutant parasites. Conclusion: PfCRT has a dual role in CQR, facilitating both efflux of harmful CQ from the DV and influx of beneficial GSH into the DV. Antioxid. Redox Signal. 19,[683][684][685][686][687][688][689][690][691][692][693][694][695]

show abstract