Polymorphisms in the Plasmodium falciparum ‘chloroquine resistance transporter’ (PfCRT) and the ‘multidrug resistance protein 1’ (PfMDR1) have been associated with changes in the parasite’s sensitivity to a broad range of drugs, including the current antimalarial amodiaquine. Both proteins are located at the membrane of the parasite’s digestive vacuole (DV) and were first identified for their roles in chloroquine resistance [1-3]. Mutant isoforms of PfCRT have been shown to transport chloroquine out of the DV (i.e., away from the drug’s site of action) [4-6]. PfMDR1 is hypothesised to pump substrates into the parasite’s DV using ATP hydrolysis, but it is currently unclear how polymorphisms in pfmdr1 influence the parasite’s response to drugs. We are investigating the contributions of PfCRT and PfMDR1 to amodiaquine resistance by expressing clinically-relevant isoforms of these proteins in the Xenopus oocyte system and measuring their capacities for amodiaquine transport. The resulting transport data is then correlated with published associations between isoforms of PfCRT and PfMDR1 and changes in (1) the parasite’s response to amodiaquine in vitro and (2) clinical outcomes of malaria patients treated with amodiaquine.
Two distinct types of mutations in PfCRT and PfMDR1 have been associated with reductions in the parasite’s sensitivity to amodiaquine. The 'GB4-type’ mutations (common in African and South-East Asian parasites) correlate with low- to moderate-levels of amodiaquine resistance, whereas the ‘7G8-type’ mutations (common in South American parasites) have been found in highly-resistant parasites. By measuring [3H]amodiaquine uptake in the Xenopus oocyte system, we have demonstrated that GB4-type and 7G8-type isoforms of PfCRT mediate amodiaquine transport, whereas the wild-type protein lacks this activity. Moreover, kinetic analyses of amodiaquine transport revealed distinct differences between the GB4-type and 7G8-type isoforms of PfCRT. We have also demonstrated amodiaquine transport via PfMDR1, and will present a model for the molecular mechanisms underlying amodiaquine resistance.