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J. Biol. Chem., Vol. 280, Issue 30, 27960-27969, July 29, 2005

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Quantitative Calcium Measurements in Subcellular Compartments of Plasmodium falciparum-infected Erythrocytes^*

Petra Rohrbach, Oliver Friedrich¶, Joachim Hentschel||, Helmut Plattner||, Rainer H. A. Fink¶, and Michael Lanzer**

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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