title: Chloroquine transport in Plasmodium falciparum. 2. Analysis of PfCRT-mediated drug transport using proteoliposomes and a fluorescent chloroquine probe.
abstract: 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 (DOI 10.1021/bi901034r ) 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 (DeltapH) and transmembrane potentials (DeltaPsi). 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 DeltaPsi or DeltapH, 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.
authors.affiliation: Department of Chemistry, Georgetown University, NW, Washington, DC 20057, USA.
authors.forename: Michelle F
authors.initials: MF
authors.lastname: Paguio
authors.forename: Mynthia
authors.initials: M
authors.lastname: Cabrera
authors.forename: Paul D
authors.initials: PD
authors.lastname: Roepe
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D001693:Biological Transport, Active / Q000502:physiology; D002384:Catalysis; D002738:Chloroquine / Q000031:analogs & derivatives* / Q000378:metabolism*; D004351:Drug Resistance; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D026901:Membrane Transport Proteins / Q000502:physiology*; D010963:Plasmodium falciparum / Q000502:physiology*; D011510:Proteolipids / Q000378:metabolism*; D015800:Protozoan Proteins / Q000502:physiology*
chemical_list: D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins; C416369:PfCRT protein, Plasmodium falciparum; D011510:Proteolipids; D015800:Protozoan Proteins; C020485:proteoliposomes; D002738:Chloroquine; D009327:4-Chloro-7-nitrobenzofurazan