PfCRT and the trans‐vacuolar proton electrochemical gradient: regulating the access of chloroquine to ferriprotoporphyrin IX

Abstract

SummaryIt is accepted that resistance of Plasmodium falciparum to chloroquine (CQ) is caused primarily by mutations in the pfcrt gene. However, a consensus has not yet been reached on the mechanism by which resistance is achieved. CQ-resistant (CQR) parasite lines accumulate less CQ than do CQsensitive (CQS) parasites. The CQR phenotype is complex with a component of reduced energydependent CQ uptake and an additional component that resembles energy-dependent CQ efflux. Here we show that the required energy input is in the form of the proton electrochemical gradient across the digestive vacuole (DV) membrane. Collapsing the DV proton gradient (or starving the parasites of glucose) results in similar levels of CQ accumulation in CQS and CQR lines. Under these conditions the accumulation of CQ is stimulated in CQR parasite lines but is reduced in CQS lines. Energy deprivation has no effect on the rate of CQ efflux from CQR lines implying that mutant PfCRT does not function as an efflux pump or active carrier. Using pfcrt-modified parasite lines we show that the entire CQ susceptibility phenotype is switched by the single K76T amino acid change in PfCRT. The efflux of CQ in CQR lines is not directly coupled to the energy supply, consistent with a model in which mutant PfCRT functions as a gated channel or pore, allowing charged CQ species to leak out of the DV.

A verapamil-sensitive chloroquine-associated H+ leak from the digestive vacuole in chloroquine-resistant malaria parasites

“…The DV localisation of PfCRT, together with the findings that the majority of the CQ accumulated by CQsensitive (CQS) parasites is within the DV (Bray et al, 2006), and that DVs isolated from CQ-resistant (CQR) parasites accumulate less CQ than those from CQS parasites (Saliba et al, 1998a), are consistent with the hypothesis that mutations in PfCRT cause CQ resistance by reducing the intravacuolar CQ concentration. How they do so is the subject of ongoing debate (Sanchez et al, 2007a).…”

“…An alternative hypothesis is that mutant PfCRT confers CQ resistance by mediating the efflux of CQ from the DV (Bray et al, 2005;Bray et al, 2006;Sanchez et al, 2007b;Warhurst et al, 2002). On the basis of experiments on intact parasitised erythrocytes, Krogstad et al proposed that CQ resistance involves an energydependent CQ efflux mechanism (Krogstad et al, 1987).…”

Stage independent chloroquine resistance and chloroquine toxicity revealed via spinning disk confocal microscopy

“…It remains reasonable to propose that a large contribution to CQR is due to disruption of toxic CQ -heme interactions within the DV during T. The disruption of CQ -heme interactions in T is thought to be due to either CQ transport from the DV [19,40], disrupted heme -Hz chemistry and heme -CQ binding due to altered DV volume and pH [28], or perhaps some combination [19,28]. Not coincidentally, current models for PfCRT include drug transport, ion transport, or both [19,20,28,40,41]. However, can these models for PfCRT explain the nearly equal levels of CQR for R and S now quantified by these bolus dose experiments ?…”

“…Taken together, this is why it is now widely assumed that CQ acts primarily on the trophozoite stage of intraerythrocytic parasites, and that this is the stage wherein the CQR mechanism must operate. Indeed, mutant PfCRT causes CQR, directly binds CQ [19] and is postulated to be a transporter that either directly or indirectly alters CQ -heme interaction within the DV during the trophozoite stage [19,20].…”

Mutation in the Plasmodium falciparum CRT Protein Determines the Stereospecific Activity of Antimalarial Cinchona Alkaloids

“…Although the endogenous function of PfCRT remains enigmatic, its localization within the DV membrane in trophozoite-stage parasites agrees with a role in drug resistance (14). Evidence suggests that mutated PfCRT, in particular a K76T mutation, confers CQ resistance by allowing enhanced efflux of CQ from the DV, thereby reducing the concentration of drug available to bind target heme (8,9,57). However, the cytotoxic properties of CQ may involve additional PfCRT-related mechanisms beyond DV accumulation (11).…”