PfCRT and the trans-vacuolar proton electrochemical gradient: regulating the access of chloroquine to ferriprotoporphyrin IX.
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.
Bray
Patrick G
PG
Department of Molecular and Biochemical Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK. p.g.bray@liv.ac.uk
Mungthin
Mathirut
M
Hastings
Ian M
IM
Biagini
Giancarlo A
GA
Saidu
Dauda K
DK
Lakshmanan
Viswanathan
V
Johnson
David J
DJ
Hughes
Ruth H
RH
Stocks
Paul A
PA
O'Neill
Paul M
PM
Fidock
David A
DA
Warhurst
David C
DC
Ward
Stephen A
SA
D000818:Animals; D002738:Chloroquine / Q000378:metabolism* / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D006427:Hemin / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000502:physiology*; D009154:Mutation / Q000235:genetics; D021261:Parasitic Sensitivity Tests; D010963:Plasmodium falciparum / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011522:Protons; D015800:Protozoan Proteins / Q000235:genetics / Q000502:physiology*; D014617:Vacuoles / Q000378:metabolism
D026901:Membrane Transport Proteins; C416369:PfCRT protein, Plasmodium falciparum; D011522:Protons; D015800:Protozoan Proteins; D006427:Hemin; D002738:Chloroquine; D005947:Glucose