Na+-dependent and Na+-independent mechanisms for inorganic phosphate uptake in Trypanosoma rangeli

Background: Trypanosoma rangeli is dependent on the presence of exogenous orthophosphate (P-i) for maximal growth and ecto-phosphatase activity is responsible for P-i supply under low P-i. Here we investigated the mechanisms of P-i uptake. Methods: We investigated the kinetics of P-32(i) transport, its Na+ and H+ dependence, its correlation with the Na+-ATPase and H+-ATPase, and gene expression of the Na+:P-i cotransporter and Na+-ATPase. Results: T. rangeli grown under limiting P-i transports this anion to the cytosol in the absence and presence of Na+, suggesting that influx is mediated by both Na+-independent and Na+-dependent transporters. Cloning studies demonstrated that this parasite expresses a P-i transporter not previously studied in trypanosomatids. The H+ ionophore, carbonylcyanide-p-trifluoromethoxyphenylhydrazone, decreased both components of P-32(i) influx by 80-95%. The H+-ATPase inhibitor, bafilomycin A(1), inhibited the Na+-independent mechanism. Furosemide, an inhibitor of ouabain-insensitive Na+-ATPase, decreased both uptake mechanisms of P-32(i) to the same extent, whereas ouabain had no effect, indicating that the former is the pump responsible for inwardly directed Na+ and the electric gradients required by the transporters. Parasite growth in high P-i had a lower P-i influx than that found in those grown in low P-i, without alteration in TrPho89 expression, showing that turnover of the transporters is stimulated by P-i starvation. Conclusions: Two modes of P-i transport, one coupled to Na+-ATPase and other coupled to H+-ATPase seem to be responsible for P-i acquisition during development of T. rangeli. General significance: This study provides the first description of the mechanism of P-i transport across the plasma membrane of trypanosomatids. (C) 2012 Elsevier B.V. All rights reserved.