Dependence of Plasmodium falciparum In Vitro Growth on the Cation Permeability of the Human Host Erythrocyte

Abstract

Intraerythrocyte growth of the malaria parasite Plasmodium falciparum induces a Ca²⁺-permeable unselective cation conductance in the host cell membrane which is inhibited by ethylisopropylamiloride (EIPA) and is paralleled by an exchange of K⁺ by Na⁺ in the host cytosol. The present study has been performed to elucidate the functional significance of the electrolyte exchange. Whole-cell patch-clamp experiments confirmed the Ca²⁺ permeability and EIPA sensitivity of the Plasmodium falciparum induced cation channel. In further experiments, ring stage-synchronized parasites were grown in vitro for 48 h in different test media. Percentage of Plasmodium-infected and phosphatidylserine-exposing erythrocytes was measured with FACS analysis by staining with the DNA-dye Syto16 and annexin V, respectively. The increase of infected cells was not significantly affected by an 8 h replacement of NaCl in the culture medium with Na-gluconate but was significantly blunted by replacement of NaCl with KCl, NMDG-Cl or raffinose. Half maximal growth was observed at about 25 mM Na⁺. The increase of infected cells was further inhibited by EIPA (IC₅₀< 10 µM) and at low extracellular free Ca²⁺. Infected cells displayed significantly stronger annexin binding, an effect mimicked by exposure of noninfected erythrocytes to oxidative stress (1 mM t-butylhydroperoxide for 15 min) or to Ca²⁺ ionophore ionomycin (1 µM for 60 min). The observations indicate that parasite growth requires the entry of both, Na⁺ and Ca²⁺ cations into the host erythrocyte probably through the EIPA sensitive cation channel. Ca²⁺ entry further induces break-down of the phospholipid asymmetry in the host membrane.