Functional characterization of the Plasmodium falciparum chloroquine-resistance transporter (PfCRT) in transformed Dictyostelium discoideum vesicles.
BACKGROUND
Chloroquine (CQ)-resistant Plasmodium falciparum malaria has been a global health catastrophe, yet much about the CQ resistance (CQR) mechanism remains unclear. Hallmarks of the CQR phenotype include reduced accumulation of protonated CQ as a weak base in the digestive vacuole of the erythrocyte-stage parasite, and chemosensitization of CQ-resistant (but not CQ-sensitive) P. falciparum by agents such as verapamil. Mutations in the P. falciparum CQR transporter (PfCRT) confer CQR; particularly important among these mutations is the charge-loss substitution K→T at position 76. Dictyostelium discoideum transformed with mutant PfCRT expresses key features of CQR including reduced drug accumulation and verapamil chemosensitization.


RESULTS
We describe the isolation and characterization of PfCRT-transformed, hematin-free vesicles from D. discoideum cells. These vesicles permit assessments of drug accumulation, pH, and membrane potential that are difficult or impossible with hematin-containing digestive vacuoles from P. falciparum-infected erythrocytes. Mutant PfCRT-transformed D. discoideum vesicles show features of the CQR phenotype, and manipulations of vesicle membrane potential by agents including ionophores produce large changes of CQ accumulation that are dissociated from vesicular pH. PfCRT in its native or mutant form blunts the ability of valinomycin to reduce CQ accumulation in transformed vesicles and decreases the ability of K(+) to reverse membrane potential hyperpolarization caused by valinomycin treatment.


CONCLUSIONS
Isolated vesicles from mutant-PfCRT-transformed D. discoideum exhibit features of the CQR phenotype, consistent with evidence that the drug resistance mechanism operates at the P. falciparum digestive vacuole membrane in malaria. Membrane potential apart from pH has a major effect on the PfCRT-mediated CQR phenotype of D. discoideum vesicles. These results support a model of PfCRT as an electrochemical potential-driven transporter in the drug/metabolite superfamily that (appropriately mutated) acts as a saturable simple carrier for the facilitated diffusion of protonated CQ.
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Janni
J
Papakrivos
Juliana M
JM
Sá
Thomas E
TE
Wellems
D000255:Adenosine Triphosphate / Q000378:metabolism; D000641:Ammonia / Q000494:pharmacology; D000818:Animals; D000962:Antimalarials / Q000494:pharmacology; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002461:Cell Line, Transformed; D002738:Chloroquine / Q000378:metabolism / Q000494:pharmacology; D022162:Cytoplasmic Vesicles / Q000187:drug effects / Q000378:metabolism*; D004023:Dictyostelium / Q000187:drug effects / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007476:Ionophores / Q000494:pharmacology; D018942:Macrolides / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D010641:Phenotype; D010963:Plasmodium falciparum / Q000187:drug effects / Q000235:genetics; D011188:Potassium / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism*; D014634:Valinomycin / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
D000962:Antimalarials; D007476:Ionophores; D018942:Macrolides; D026901:Membrane Transport Proteins; C416369:PfCRT protein, Plasmodium falciparum; D015800:Protozoan Proteins; D014634:Valinomycin; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000641:Ammonia; C036978:concanamycin A; D002738:Chloroquine; D000255:Adenosine Triphosphate; D014700:Verapamil; D011188:Potassium