Vacuolar-type H+-ATPase regulates cytoplasmic pH in Toxoplasma gondii tachyzoites

Abstract

Cytoplasmic pH (pH_i) regulation was studied in Toxoplasma gondii tachyzoites by using the fluorescent dye 2ʹ,7ʹ-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. 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 low concentrations of N,Nʹ-dicyclohexylcarbodi-imide (DCCD), N-ethylmaleimide (NEM) or bafilomycin A₁. Bafilomycin A₁ also inhibited the recovery of tachyzoite pH_i after an acid load with sodium propionate. Similar concentrations of DCCD, NEM and bafilomycin A₁ produced depolarization of the plasma membrane potential as measured with bis-(1,3-diethylthiobarbituric)trimethineoxonol (bisoxonol), and DCCD prevented the hyperpolarization that accompanies acid extrusion after the addition of propionate, in agreement with the electrogenic nature of this pump. Confocal laser scanning microscopy indicated that, in addition to 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 biotinylation of cell surface proteins and immunoprecipitation with antibodies against V-H⁺-ATPases. Taken together, the results are consistent with the presence of a functional V-H⁺-ATPase in the plasma membrane of these intracellular parasites and with an important role of this enzyme in the regulation of pH_i homoeostasis in these cells.