title: A kinetic fluorescence assay reveals unusual features of Ca⁺⁺ uptake in Plasmodium falciparum-infected erythrocytes.
abstract: BACKGROUND
  To 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.
  
  
  METHODS
  Based 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.
  
  
  RESULTS
  Both 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++.
  
  
  CONCLUSIONS
  Nondestructive 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.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Zipprer
authors.forename: McKinzie
authors.initials: M
authors.lastname: Neggers
authors.forename: Ambuj
authors.initials: A
authors.lastname: Kushwaha
authors.forename: Kempaiah
authors.initials: K
authors.lastname: Rayavara
authors.affiliation: The Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. sdesai@niaid.nih.gov.
authors.forename: Sanjay A
authors.initials: SA
authors.lastname: Desai
mesh_terms: D000814:Aniline Compounds / Q000032:analysis; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000737:chemistry; D004912:Erythrocytes / Q000378:metabolism* / Q000469:parasitology*; D005453:Fluorescence; D006801:Humans; D010963:Plasmodium falciparum / Q000254:growth & development*; D013194:Staining and Labeling / Q000379:methods; D014966:Xanthenes / Q000032:analysis
chemical_list: D000814:Aniline Compounds; D014966:Xanthenes; C561295:fluo-8 AM; D002118:Calcium