Vacuolar H+-ATPase localized in plasma membranes of malaria parasite cells, Plasmodium falciparum, is involved in regional acidification of parasitized erythrocytes

Recent biochemical studies involving 2',7'-bis-(2-carboxyethyl)-5,6 -carbox ylfluorescein (BCECF)-labeled saponin-permeabilized and parasitized erythro cytes indicated that malaria parasite cells maintain the resting cytoplasmi c pH at about 7.3, and treatment with vacuolar proton-pump inhibitors reduc es the resting pH to 6.7, suggesting proton extrusion from the parasite cel ls via vacuolar H+-ATPase (Saliba, K. J., and Kirk, K. (1999) J, Biol. Chem , 274, 33213-33219), In the present study, we investigated the localization of vacuolar H+-ATPase in Plasmodium falciparum cells infecting erythrocyte s, Antibodies against vacuolar H+-ATPase subunit A and B specifically immun ostained the infecting parasite cells and recognized a single 67- and 55-kD a polypeptide, respectively. Immunoelectron microscopy indicated that the i mmunological counterpart of V-ATPase subunits A and B is localized at the p lasma membrane, small clear vesicles, and food vacuoles, a lower extent bei ng detected at the parasitophorus vacuolar membrane of the parasite cells. We measured the cytoplasmic pH of both infected erythrocytes and invading m alaria parasite cells by microfluorimetry using BCECF fluorescence. It was found that a restricted area of the erythrocyte cytoplasm near a parasite c ell is slightly acidic, being about pH 6.9. The pH increased to pH 7.3 upon the addition of either concanamycin B or bafilomycin A(1), specific inhibi tors of vacuolar H+-ATPase. Simultaneously, the cytoplasmic pH of the infec ting parasite cell decreased from pH 7.3 to 7.1. Neither vanadate at 0.5 mM , an inhibitor of P-type H+-ATPase, nor ethylisopropylamiloride at 0.2 mM, an inhibitor of Na+/H+-exchanger, affected the cytoplasmic pH of erythrocyt es or infecting parasite cells. These results constitute direct evidence th at plasma membrane vacuolar H+-ATPase is responsible for active exclusion o f protons from the parasite cells.