Biochemical characterization and chemical inhibition of PfATP4-associated Na+-ATPase activity in Plasmodium falciparum membranes

Abstract

The antimalarial activity of chemically diverse compounds, including the clinical candidate cipargamin, has been linked to the ATPase PfATP4 in the malaria-causing parasite Plasmodium falciparum The characterization of PfATP4 has been hampered by the inability thus far to achieve its functional expression in a heterologous system. Here, we optimized a membrane ATPase assay to probe the function of PfATP4 and its chemical sensitivity. We found that cipargamin inhibited the Na⁺-dependent ATPase activity present in P. falciparum membranes from WT parasites and that its potency was reduced in cipargamin-resistant PfATP4-mutant parasites. The cipargamin-sensitive fraction of membrane ATPase activity was inhibited by all 28 of the compounds in the "Malaria Box" shown previously to disrupt ion regulation in P. falciparum in a cipargamin-like manner. This is consistent with PfATP4 being the direct target of these compounds. Characterization of the cipargamin-sensitive ATPase activity yielded data consistent with PfATP4 being a Na⁺ transporter that is sensitive to physiologically relevant perturbations of pH, but not of [K⁺] or [Ca²⁺]. With an apparent K_m for ATP of 0.2 mm and an apparent K_m for Na⁺ of 16-17 mm, the protein is predicted to operate at below its half-maximal rate under normal physiological conditions, allowing the rate of Na⁺ efflux to increase in response to an increase in cytosolic [Na⁺]. In membranes from a cipargamin-resistant PfATP4-mutant line, the apparent K_m for Na⁺ is slightly elevated. Our study provides new insights into the biochemical properties and chemical sensitivity of an important new antimalarial drug target.

Keywords: ATPase; PfATP4; Plasmodium falciparum; antiplasmodial drug; cipargamin; drug action; drug resistance; malaria; membrane transport; sodium transporter.