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

Abstract

It 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 CQ-sensitive (CQS) parasites. The CQR phenotype is complex with a component of reduced energy-dependent 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.

Fig. 1

The effect of glucose starvation on the accumulation of [³H]-CQ by CQS and CQR lines of P. falciparum. Parasite lines were incubated with or without 10 mM glucose and CQ accumulation was measured over a 10 min period using a concentration of 2 nM [³H]-CQ. The CQ cellular accumulation ratio (CAR) is the ratio of the intracellular versus the extracellular [³H]-CQ concentration, [CQ]_in/[CQ]_out, over a 10 min period. A. Data for the non-transformed Dd2 (CQR) and GC03 (CQS) lines. B. Data for the C2^GC03 and C3^Dd2 lines (produced by allelic modification of the pfcrt locus in the GC03 line) that express the GC03 wild-type CQS and the Dd2 mutant CQR pfcrt alleles respectively (Sidhu et al., 2002). C. Data for the C-1^Dd2 and T76K-1^Dd2 recombinant clones (produced by allelic modification of the pfcrt locus in Dd2 parasites), with C-1^Dd2 expressing the parental Dd2 allele whereas T76K-1^Dd2 expresses a variant Dd2 ‘back-mutant’ allele in which the mutant threonine codon 76 (T76) was replaced with the wild-type lysine codon 76 (K76) (Lakshmanan et al., 2005). Histogram bars represent means ± standard deviation of at least five independent experiments, each performed in triplicate. The effect of glucose starvation was significant in all cases (Mann–Whitney, P < 0.001).

Fig. 2

The effect of FCCP on the accumulation of [³H]-CQ by CQS and CQR lines of P. falciparum. Parasite lines were incubated with 10 mM glucose, with or without 10 μM FCCP and CQ accumulation was measured over a 10 min period using a concentration of 2 nM [³H]-CQ. For all experiments, CQ cellular accumulation ratio (CAR) is given as the ratio of the intracellular versus the extracellular [³H]-CQ concentration, [CQ]_in/[CQ]_out, over a 10 min period. A. Data for the non-transformed Dd2 and GC03 lines. B. Data for the C2^GC03 and C3^Dd2 lines. C. Data for the C-1^Dd2 and T76K-1^Dd2 recombinant clones. Histogram bars represent means ± standard deviation of at least seven independent experiments, each performed in triplicate. The effect of FCCP was significant in all cases (Mann–Whitney, P < 0.001).

Fig. 3

The DV is the major CQ accumulating compartment in P. falciparum. (A and B) Data for the C2^GC03 and C3^Dd2 lines. In these experiments, parasite suspensions were incubated with 2 nM [³H]-CQ, with or without 10 μM VP or 10 μM FCCP for a 10 min period prior to subcellular fractionation. Histogram bars represent means ± standard deviation of at least eight independent experiments, each performed in duplicate. The effect of FCCP was significant in all cases (Mann–Whitney, P < 0.001). The effect of VP was significant only in the CQR line (Mann–Whitney, P < 0.001).

Fig. 4

A pre-loading step is required for the trans-stimulation effect. A. Data for the C3^Dd2 line. Accumulation of [³H]-CQ (1 nM) was measured after pre-loading with unlabelled CQ and washing as described (Sanchez et al., 2003). B. Data for the same C3^Dd2 line, this time with the pre-loading step omitted and the labelled and unlabelled CQ mixed prior to incubation with parasites. C. Data for various transformed and non-transformed CQR lines, obtained using the same protocol as in (B). For all experiments, [³H]-CQ accumulation was measured after 4 min of incubation (given as the ratio of the intracellular versus the extracellular [³H]-CQ concentration, [CQ]_in/[CQ]_out, at 4 min). Accumulation of label was plotted as a function of the pre-equilibrium unlabelled CQ concentration. Uptake studies were performed in medium containing 10 mM glucose. Each data point represents the mean ± standard deviation of duplicate observations from at least four independent experiments.

Fig. 5

Efflux of CQ, effect of other haem-binding drugs and effect of energy starvation. A. Data for the CQS GC03 line measured in the presence or absence of 200 nM HF or 10 μM PQ or 10 μM VP. B. Data for the Dd2 CQR line measured in the presence or absence of 10 mM glucose or 10 mM glucose with or without 10 μM FCCP or 10 mM glucose with or without 5 μM VP. C. Data for the GC03 CQS line measured in the presence or absence of 10 mM glucose or 10 mM glucose with or without 10 μM FCCP. Data points are means of duplicate observations from at least two independent experiments.

Fig. 6

Influence of energy starvation on CQ efflux in lines harbouring wild-type and mutant alleles of pfcrt. CQ efflux was measured in the presence or absence of 10 mM glucose or 10 mM glucose with or without 10 μM FCCP. A and B. Data for the C2^GC03 and C3^Dd2 lines respectively. C and D. Data for the T76K-1^Dd2 and C-1^Dd2 recombinant clones respectively. Data points are means of duplicate observations from at least two independent experiments.