Development of fluorescence-based assays to determine changes in membrane potential and K+ levels in isolated Plasmodium falciparum parasites

Abstract

While malaria is a treatable disease caused by Plasmodium parasites, resulting in worldwide mortality. Plasmodium falciparum parasites are vulnerable to changes in intracellular ion concentrations. The parasite Na+/H+-ATPase (PfATP4) responsible for the simultaneous efflux of Na+ and the influx of H+ is targeted by chemically diverse antiplasmodial compounds, while triaminopyrimidine resistance is associated with mutations in a V-type H+-ATPase. In P. falciparum parasites, a high intracellular K+ concentration is maintained by the influx of K+ through K+ channels, against the concentration gradient due to a highly negative membrane potential. Given that K+ is the most abundant intracellular cation, it is possible that changing intracellular K+ levels would prevent parasite proliferation. Any changes in the intracellular K+ also affect the membrane potential, ultimately leading to cell death. Previous whole-cell proliferation assays showed that putative K+ channel inhibitors and ionophores inhibit proliferation, but these studies did not test the antiproliferative effects with changes in intracellular K+. We found that 250 nM DiBAC4(3) provided a high fluorescent signal in isolated asexual P. falciparum trophozoites after 30 min incubation. This condition resulted in a signal-to-noise of 119.28 and a Z’-factor of 0.83 and was used for further analysis of changes in the parasite’s membrane potential after treatment with inhibitors. APG-1 (5 μM) resulted in the highest signal-to-background ratio after 60 minutes, that also resulted in high signal-to-noise ratios (276.26) and a Z’-factor of 0.89. Therefore, the two fluorescent probes could successfully be detected in P. falciparum parasites and subsequently evaluate changes in Δψ and intracellular K+.