title: Diverse mutational pathways converge on saturable chloroquine transport via the malaria parasite's chloroquine resistance transporter.
abstract: Mutations in the chloroquine resistance transporter (PfCRT) are the primary determinant of chloroquine (CQ) resistance in the malaria parasite Plasmodium falciparum. A number of distinct PfCRT haplotypes, containing between 4 and 10 mutations, have given rise to CQ resistance in different parts of the world. Here we present a detailed molecular analysis of the number of mutations (and the order of addition) required to confer CQ transport activity upon the PfCRT as well as a kinetic characterization of diverse forms of PfCRT. We measured the ability of more than 100 variants of PfCRT to transport CQ when expressed at the surface of Xenopus laevis oocytes. Multiple mutational pathways led to saturable CQ transport via PfCRT, but these could be separated into two main lineages. Moreover, the attainment of full activity followed a rigid process in which mutations had to be added in a specific order to avoid reductions in CQ transport activity. A minimum of two mutations sufficed for (low) CQ transport activity, and as few as four conferred full activity. The finding that diverse PfCRT variants are all limited in their capacity to transport CQ suggests that resistance could be overcome by reoptimizing the CQ dosage.
authors.affiliation: Research School of Biology, The Australian National University, Canberra, ACT 0200, Australia.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Summers
authors.forename: Anurag
authors.initials: A
authors.lastname: Dave
authors.forename: Tegan J
authors.initials: TJ
authors.lastname: Dolstra
authors.forename: Sebastiano
authors.initials: S
authors.lastname: Bellanca
authors.forename: Rosa V
authors.initials: RV
authors.lastname: Marchetti
authors.forename: Megan N
authors.initials: MN
authors.lastname: Nash
authors.forename: Sashika N
authors.initials: SN
authors.lastname: Richards
authors.forename: Valerie
authors.initials: V
authors.lastname: Goh
authors.forename: Robyn L
authors.initials: RL
authors.lastname: Schenk
authors.forename: Wilfred D
authors.initials: WD
authors.lastname: Stein
authors.forename: Kiaran
authors.initials: K
authors.lastname: Kirk
authors.forename: Cecilia P
authors.initials: CP
authors.lastname: Sanchez
authors.forename: Michael
authors.initials: M
authors.lastname: Lanzer
authors.forename: Rowena E
authors.initials: RE
authors.lastname: Martin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002738:Chloroquine / Q000378:metabolism*; D004351:Drug Resistance; D006239:Haplotypes; D007700:Kinetics; D016778:Malaria, Falciparum / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D009865:Oocytes; D010271:Parasites / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000378:metabolism*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection; D014982:Xenopus laevis
chemical_list: D026901:Membrane Transport Proteins; D050505:Mutant Proteins; C416369:PfCRT protein, Plasmodium falciparum; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D002738:Chloroquine
keywords: Xenopus oocytes; drug resistance; evolutionary biochemistry
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