FIG 4.

Resting cytosolic pH of orthologue replacement lines and the concentration-dependent increase in pH after addition of cipargamin. Trophozoite-stage parasites were loaded with the pH-sensitive fluorophore BCECF, and the fluorescence of the suspensions was measured in a spectrophotometer. (A) Addition of 100 nM concanamycin A, a V-type H⁺-ATPase inhibitor, caused a rapid acidification of the pH_i for the P. knowlesi parental line as shown before in P. falciparum (35). Shown here is a trace from a single experiment, but it is representative of results obtained on multiple occasions and in all lines. (B) Dose-dependent effect of adding the ATP4 inhibitor cipargamin on alkalinization of pH_i in the P. knowlesi parental line. Data shown are the average from at least 3 independent biological replicates ± SEM (C) Measurements of baseline pH_i show no significant difference among the P. knowlesi parental parasites and the orthologue replacement lines (pH_i ~7.25). The P. falciparum intracellular pH was slightly higher (7.45). (D) We determined the fold increase in pH_i after the addition of 100 nM cipargamin to the parasite lines. The fold increase was determined by dividing the pH_i at 10 min by the resting pH_i before the addition of cipargamin (t = 0 min). Data for the resting pH experiments are from technical duplicate experiments performed with at least five biological repeats (up to 10 times). Data from the fold change in pH experiments are from duplicate experiments performed on two to three separate occasions. ns, non significant (P > 0.05).