FIG 3.

Resting intracellular sodium concentration ([Na⁺]_i) of the orthologue replacement lines and the concentration-dependent increase in [Na⁺]_i after addition of cipargamin. Trophozoite-stage parasites were loaded with the Na⁺-sensitive fluorophore SBFI, and the fluorescence of the suspensions was measured in a spectrophotometer. (A) Measurements of resting [Na⁺]_i show values of between 6.34 mM for the P. falciparum 3D7 line (gray square) and 22.03 mM for the PocATP4^OR line (red circle). However, there was no significant difference for any of the lines compared to the PfATP4^OR line (green circle; P > 0.0681). Data show the mean ± SEM. (B) Addition of cipargamin resulted in an immediate dose-dependent increase in the resting [Na⁺]_i inside all parasite lines. Shown here are representative traces from a single experiment demonstrating the impact of adding 20 nM cipargamin (green trace) or 100 nM cipargamin (blue trace) on the resting [Na⁺]_i of the PocATP4^OR line. (C) Addition of 100 nM cipargamin to the parasite lines results in an increase in the [Na⁺]_i from steady state. We determined the initial rate of increase in the [Na⁺]_i by fitting a linear regression curve to the [Na⁺]_i increase data across that 10-min exposure. The slope of that curve provides the rate of increase (in micromolar concentration per second). Experiments for resting [Na⁺] were performed in technical duplicates for at least three biological repeats, but some were performed to eight times. Experiments for the initial rate of increase in [Na⁺] were performed in technical duplicates for at least three biological repeats, but some were performed up to five times. ***, P ≤ 0.001; ns, non significant (P > 0.05).