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J. Biol. Chem., Vol. 280, Issue 30, 27960-27969, July 29, 2005
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¶
**
From the
Hygiene Institut, Abteilung Parasitologie, Universitätsklinikum Heidelberg, Im Neuenheimer Feld 324, D-69120 Heidelberg, the ¶Medical Biophysics, Institute of Physiology and Pathophysiology, University of Heidelberg, Im Neuenheimer Feld 326, D-69120 Heidelberg, and the ||Department of Biology, University of Konstanz, D-78457 Konstanz, Germany
The acidic food vacuole exerts several important functions during intraerythrocytic development of the human malarial parasite Plasmodium falciparum. Hemoglobin taken up from the host erythrocyte is degraded in the food vacuole, and the heme liberated during this process is crystallized to inert hemozoin. Several anti-malarial drugs target food vacuolar pathways, such as hemoglobin degradation and heme crystallization. Resistance and sensitization to some antimalarials is associated with mutations in food vacuolar membrane proteins. Other studies suggest a role of the food vacuole in ion homeostasis, and release of Ca2+ from the food vacuole may mediate adopted physiological responses. To investigate whether the food vacuole is an intracellular Ca2+ store, which in turn may affect other physiological functions in which this organelle partakes, we have investigated total and exchangeable Ca2+ within the parasite's food vacuole using x-ray microanalysis and quantitative confocal live cell Ca2+ imaging. Apparent free Ca2+ concentrations of
90,
350, and
400 nM were found in the host erythrocyte cytosol, the parasite cytoplasm, and the food vacuole, respectively. In our efforts to determine free intracellular Ca2+ concentrations, we evaluated several Ca2+-sensitive fluorochromes in a live cell confocal setting. We found that the ratiometric Ca2+ indicator Fura-Red provides reliable determinations, whereas measurements using the frequently used Fluo-4 are compromised due to problems arising from phototoxicity, photobleaching, and the strong pH dependence of the dye. Our data suggest that the food vacuole contains only moderate amounts of Ca2+, disfavoring a role as a major intracellular Ca2+ store.
Received for publication, January 21, 2005 , and in revised form, May 24, 2005.
* This work was supported by the Landesstiftung Baden-Württemberg. 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.
Both authors contributed equally to this study.
** To whom correspondence should be addressed. Tel.: 49-6221-567845; Fax: 49-6221-564643; E-mail: michael_lanzer{at}med.uni-heidelberg.de.
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