Chloroquine Transport inPlasmodium 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 [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.

Functional Comparison of 45 Naturally Occurring Isoforms of the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT)

“…Thus, the orientation of thermodynamic driving forces for transport versus PfCRT membrane topology is preserved. Because CQ 2+ transport by PfCRT is stimulated by ΔΨ and ΔpH oriented as in the DV, 7,9,23 in yeast, PfCRT mediates fast downhill transport of CQ 2+ from outside the cell (positive membrane potential, low pH) toward the yeast cytosol (negative potential, high pH) (Figure 1B; see also ref 9). The inward transport of toxic CQ by PfCRT expressed in the yeast leads to decreased rates of yeast growth relative to control.…”

“…5,6 Mutant, CQR-associated isoforms of PfCRT are believed to confer CQR by mediating an increased level of transport of charged CQ out of the DV down its electrochemical gradient and by decreasing the number of CQ–heme target interactions via perturbations in DV volume and/or pH. 1–3,5,7 …”

A High-Content Phenotypic Screen Reveals the Disruptive Potency of Quinacrine and 3′,4′-Dichlorobenzamil on the Digestive Vacuole of Plasmodium falciparum

“…These mutations facilitate the efflux of CQ from the DV, resulting in lower intravacuolar concentrations of CQ (20)(21)(22)(23). This would explain the lowered ability of CQ to induce Ca 2ϩ redistribution in 7G8 and K1 ( Fig.…”

Glutathione Transport: A New Role for PfCRT in Chloroquine Resistance

“…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.…”