A kinetic fluorescence assay reveals unusual features of Ca++ uptake in Plasmodium falciparum-infected erythrocytes

Malaria Journal

Research

Elizabeth M Zipprer¹  Kempaiah Rayavara¹  Sanjay A Desai¹  McKinzie Neggers¹  Ambuj Kushwaha¹ 

[1] The Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 20892, Bethesda, MD, USA;

关键词: Calcium channels;    Malaria parasites;    Human erythrocytes;    Fluo-8;    Fluorescence kinetics;    Antimalarial drug discovery;   

DOI  :  10.1186/1475-2875-13-184

 received in 2013-09-02, accepted in 2014-05-11,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundTo facilitate development within erythrocytes, malaria parasites increase their host cell uptake of diverse solutes including Ca++. The mechanism and molecular basis of increased Ca++ permeability remains less well studied than that of other solutes.MethodsBased on an appropriate Ca++ affinity and its greater brightness than related fluorophores, Fluo-8 was selected and used to develop a robust fluorescence-based assay for Ca++ uptake by human erythrocytes infected with Plasmodium falciparum.ResultsBoth uninfected and infected cells exhibited a large Ca++-dependent fluorescence signal after loading with the Fluo-8 dye. Probenecid, an inhibitor of erythrocyte organic anion transporters, abolished the fluorescence signal in uninfected cells; in infected cells, this agent increased fluorescence via mechanisms that depend on parasite genotype. Kinetic fluorescence measurements in 384-well microplates revealed that the infected cell Ca++ uptake is not mediated by the plasmodial surface anion channel (PSAC), a parasite nutrient channel at the host membrane; it also appears to be distinct from mammalian Ca++ channels. Imaging studies confirmed a low intracellular Ca++ in uninfected cells and higher levels in both the host and parasite compartments of infected cells. Parasite growth inhibition studies revealed a conserved requirement for extracellular Ca++.ConclusionsNondestructive loading of Fluo-8 into human erythrocytes permits measurement of Ca++ uptake kinetics. The greater Ca++ permeability of cells infected with malaria parasites is apparent when probenecid is used to inhibit Fluo-8 efflux at the host membrane. This permeability is mediated by a distinct pathway and may be essential for intracellular parasite development. The miniaturized assay presented here should help clarify the precise transport mechanism and may identify inhibitors suitable for antimalarial drug development.

【 授权许可】

Unknown   
© Zipprer et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.