title: Monitoring PfMDR1 transport in Plasmodium falciparum.
abstract: BACKGROUND
  The Plasmodium falciparum multidrug resistance 1 transporter, PfMDR1, contains five amino acid polymorphisms that are suggested to be involved in altered drug transport from the parasite's cytosol into the digestive vacuole (DV). Transport of a substrate into another intracellular compartment influences drug availability at its site of action, therefore making the parasite more susceptible or resistant to a drug. Fluo-4 is a known fluorescent substrate that can be used as a molecular tool to investigate transport dynamics of PfMDR1 in many parasite strains.
  
  
  METHODS
  Six P. falciparum strains with varying PfMDR1 mutations were loaded with Fluo-4 AM. Accumulation of the fluorophore in the DV was measured using confocal microscopy. The role of a key amino acid mutation was verified using selected parasite clones with point mutations at PfMDR1 amino acid position 1042. Equal expression of PfMDR1 was confirmed by Western blot.
  
  
  RESULTS
  Fluo-4 was transported by PfMDR1 into the DV of most drug-sensitive and -resistant parasites. Asparagine at PfMDR1 amino acid position 1042 was crucial for Fluo-4 transport, while the N1042D substitution abolished Fluo-4 transport. Competition studies of Fluo-4 with chloroquine, quinine and mefloquine were performed on parasites harbouring asparagine at position 1042. A distinct Fluo-4 transport inhibition pattern for each tested anti-malarial drug was observed in parasite strains of different genetic background.
  
  
  CONCLUSIONS
  This study demonstrates that Fluo-4 can be used to investigate PfMDR1 transport dynamics in both drug-sensitive and -resistant parasites. Furthermore, direct evidence of altered Fluo-4 transport in PfMDR1 is linked to a single amino acid mutation in the substrate binding pocket. This system offers a great tool to investigate the role of substrate transport by PfMDR1 and the mutations necessary to support transport, which would lead to new insights for the development of novel anti-malarial drugs.
authors.affiliation: Institute of Parasitology, McGill University, Ste. Anne de Bellevue (Montreal), QC, H9X-3V9, Canada. sarah.reiling@mail.mcgill.ca.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Reiling
authors.affiliation: Institute of Parasitology, McGill University, Ste. Anne de Bellevue (Montreal), QC, H9X-3V9, Canada. petra.rohrbach@mcgill.ca.
authors.forename: Petra
authors.initials: P
authors.lastname: Rohrbach
mesh_terms: D000814:Aniline Compounds / Q000378:metabolism*; D001692:Biological Transport; D004351:Drug Resistance; D005456:Fluorescent Dyes / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010963:Plasmodium falciparum / Q000235:genetics / Q000378:metabolism*; D011110:Polymorphism, Genetic; D014966:Xanthenes / Q000378:metabolism*
chemical_list: D000814:Aniline Compounds; D005456:Fluorescent Dyes; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D014966:Xanthenes; C409648:Fluo 4; C513498:Mdr1 protein, Plasmodium falciparum