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J. Biol. Chem., Vol. 280, Issue 27, 25596-25603, July 8, 2005

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Dictyostelium discoideum Expresses a Malaria Chloroquine Resistance Mechanism upon Transfection with Mutant, but Not Wild-type, Plasmodium falciparum Transporter PfCRT^*

Bronwen Naudé, Joseph A. Brzostowski¶||, Alan R. Kimmel¶, and Thomas E. Wellems**

From the Laboratory of Malaria and Vector Research, NIAID, National Institutes of Health, Bethesda, Maryland 20892-8132 and the ^¶Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-8028

Chloroquine resistance in Plasmodium falciparum malaria results from mutations in PfCRT, a member of a unique family of transporters present in apicomplexan parasites and Dictyostelium discoideum. Mechanisms that have been proposed to explain chloroquine resistance are difficult to evaluate within malaria parasites. Here we report on the targeted expression of wild-type and mutant forms of PfCRT to acidic vesicles in D. discoideum. We show that wild-type PfCRT has minimal effect on the accumulation of chloroquine by D. discoideum, whereas forms of PfCRT carrying a key charge-loss mutation of lysine 76 (e.g. K76T) enable D. discoideum to expel chloroquine. As in P. falciparum, the chloroquine resistance phenotype conferred on transformed D. discoideum can be reversed by the channel-blocking agent verapamil. Although intravesicular pH levels in D. discoideum show small acidic changes with the expression of different forms of PfCRT, these changes would tend to promote intravesicular trapping of chloroquine (a weak base) and do not account for reduced drug accumulation in transformed D. discoideum. Our results instead support outward-directed chloroquine efflux for the mechanism of chloroquine resistance by mutant PfCRT. This mechanism shows structural specificity as D. discoideum transformants that expel chloroquine do not expel piperaquine, a bisquinoline analog of chloroquine used frequently against chloroquine-resistant parasites in Southeast Asia. PfCRT, nevertheless, may have some ability to act on quinine and quinidine. Transformed D. discoideum will be useful for further studies of the chloroquine resistance mechanism and may assist in the development and evaluation of new antimalarial drugs.

Received for publication, March 23, 2005 , and in revised form, May 5, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Division of Pharmacology, University of Cape Town, South Africa.

^|| Present address: Laboratory of Immunogenetics, NIAID, National Institutes of Health, Rockville, Maryland 20852.

^** To whom correspondence should be addressed. E-mail: twellems{at}niaid.nih.gov.

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