Molecular cloning and characterization of ouabain-insensitive Na+-ATPase in the parasitic protist, Trypanosoma cruzi

Maintaining low intracellular sodium concentrations is vital for almost all organisms. Na+ efflux is generally governed by P-type ATPases, Na+/K+-ATPase in animals and Na+-ATPase, called ENA, in fungi and plants. Trypanosoma cruzi, which parasitizes mammalian cells, must undergo drastic adaptations to high Na+ concentrations outside and low Na+ concentrations inside host cells. However, T. cruzi Na+ efflux pumps have not been identified. We report here the cloning and characterization of the gene encoding Na+-ATPase in T cruzi, which resembled fungal and plant ENAs, termed TcENA. TcENA was a plasma membrane protein expressed throughout the parasite life cycle. The transcription level of TcENA was higher in insect stage epimastigotes and blood stream trypomastigotes than in intracellular amastigotes, probably reflecting the high Na+ concentration outside the host cells. Biochemical analysis of TcENA expressed heterologously in mammalian cells demonstrated, for the fist time, that the ATPase activity of TcENA is stimulated by both Na+ and K+ and is insensitive to ouabain, a specific inhibitor of Na+/K+-ATPases. Furthermore, epimastigotes overproducing TcENA showed increased tolerance to high Na+ stress. Our findings suggest that TcENA acts as a sodium pump and provide insights into the regulation of ion homeostasis in the parasitic protist.