VACUOLAR-TYPE H-ATPASE REGULATES CYTOPLASMIC PH IN TOXOPLASMA-GONDII TACHYZOITES()

Cytoplasmic pH (pH(i)) regulation was studied in Toxoplasma gondii tac hyzoites by using the fluorescent dye ',7'-bis-(2-carboxyethyl)-5(6)-c arboxyfluorescein. Their mean baseline pH(i) (7.07+/-0.06; n = 5) was not significantly affected in the absence of extracellular Na+, K+ or HCO3- but was significantly decreased in a dose-dependent manner by lo w concentrations of N,N'-dicyclohexylcarbodi-imide (DCCD), N-ethylmale imide (NEM) or bafilomycin A(1). Bafilomycin A(1) also inhibited the r ecovery of tachyzoite pH(i) after an acid load with sodium propionate. Similar concentrations of DCCD, NEM and bafilomycin A(1) produced dep olarization of the plasma membrane potential as measured with bis-(1,3 -diethylthiobarbituric)trimethineoxonol (bisoxonol), and DCCD prevente d the hyperpolarization that accompanies acid extrusion after the addi tion of propionate, in agreement with the electrogenic nature of this pump. Confocal laser scanning microscopy indicated that, in addition t o being located in cytoplasmic vacuoles, the vacuolar (V)-H+-ATPase of T. gondii tachyzoites is also located in the plasma membrane. Surface localization of the V-H+-ATPase was confirmed by experiments using bi otinylation of cell surface proteins and immunoprecipitation with anti bodies against V-H+-ATPases. Taken together, the results are consisten t with the presence of a functional V-H+-ATPase in the plasma membrane of these intracellular parasites and with an important role of this e nzyme in the regulation of pH(i) homoeostasis in these cells.