Proton and sodium pumps regulate the plasma membrane potential of different stages of Trypanosoma cruzi

Abstract

We studied the plasma membrane potential (DeltaPsi) in different stages of Trypanosoma cruzi using the potentiometric fluorescent dye bisoxonol. Our results clearly demonstrate that a proton pump plays a significant role in the regulation of DeltaPsi in all stages of T. cruzi as evidenced by depolarization of the DeltaPsi by H(+)-ATPase inhibitors dicyclohexylcarbo-diimide, N-ethylmaleimide, diethylstilbestrol, and bafilomycin A(1). The H(+)-ATPase appeared to be activated by acidic conditions in trypomastigotes as evidenced by hyperpolarization of the DeltaPsi upon addition of acid. In contrast to epimastigotes and amastigotes, the DeltaPsi of trypomastigotes was markedly sensitive to extracellular Na(+) and K(+) concentrations and evidence was provided for an outward directed Cl(minus sign) channel in this stage as well. Additionally, for the first time, the existence of an ouabain-sensitive functional sodium pump was demonstrated in T. cruzi based on the depolarization of the DeltaPsi in trypomastigotes by ouabain in the presence of Na(+). In the epimastigotes, ouabain had no effect on the DeltaPsi in a sodium rich buffer. However, ouabain induced an additional significant depolarization in these stages when their DeltaPsi was already partially depolarized by the H(+)-ATPase inhibitor dicyclohexylcarbo-diimide, supporting the presence of an ouabain-sensitive sodium pump whose activity is masked by the H(+)-ATPase. In the amastigotes no evidence for a functional sodium pump could be found. In support of an inwardly directed K(+) channel, the DeltaPsi was hyperpolarized by K(+) free buffer in trypomastigotes and epimastigotes and by Ba(2+) in epimastigotes and amastigotes. The presence of K(+) channels in amastigotes and a sodium pump in trypomastigotes, in addition to the H(+)-ATPase, could provide important new targets for trypanocidal drug development.