Na⁺ regulation in the intraerythrocytic malaria parasite

Abstract

The maintenance of a low intracellular [Na⁺] ([Na⁺]i) is a crucial aspect of cellular physiology. In mammalian cells this is achieved through the extrusion of Na⁺ via the well-characterised Na⁺/K⁺-ATPase. Approximately 12 hr after invasion of the human erythrocyte by the malaria parasite there is a profound increase in the permeability of the erythrocyte membrane to a wide range of solutes, including Na⁺. Na⁺ enters the infected erythrocyte via parasite-induced 'New Permeability Pathways' and there is, as a result, an increase in [Na⁺] in the erythrocyte compartment, with [Na⁺]i eventually reaching levels similar to those in the extra erythrocytic plasma (~130 mM). The parasite itself maintains a low [Na⁺]i. The resulting large inwardly-directed electrochemical Na⁺ gradient across the parasite plasma membrane energises the accumulation within the parasite of at least one essential nutrient (inorganic phosphate). The aim of this thesis was to characterise the mechanisms involved in Na⁺ regulation in the mature asexual 'trophozoite' stage of the human malaria parasite Plasmodium falciparum. The Na⁺-sensitive, fluorescent dye Sodium-binding BenzoFuran Isophthalate (SBFI) was used to measure [Na⁺]i in parasites functionally isolated from their host cells by saponin-permeabilisation of the host erythrocyte membrane. Under physiologically relevant conditions the resting [Na⁺]i in isolated trophozoites was estimated to be ~11 mM. Maintenance of [Na⁺]i was sensitive to the P-type ATPase inhibitor orthovanadate, consistent with Na⁺ extrusion being via a P-type Na⁺-ATPase, similar to the ENA (exitus natru; exit of sodium)-type ATPases that operate in some other protozoa, fungi and lower plants. ENA ATPases have been predicted to antiport H⁺ and the data obtained here are consistent with this being true of the P. falciparum Na⁺ extrusion system. The P. falciparum genome encodes a number of putative P-type ATPases; one of these, PfATP4, was found to share significant sequence similarities to ENA ATPases of other protozoa. A recent study showed that mutations in PfATP4 confer resistance to a newly-described class of antimalarials, the spiroindolones. The effect of the spiroindolones on ion regulation was therefore investigated. Several spiroindolones were shown to cause a profound disruption of [Na⁺]<subscript>i regulation. In parasites with mutant PfATP4 there was both an impairment of Na⁺ regulation and a decrease in the spiroindolone-sensitivity of Na⁺ regulation. These results are consistent with PfATP4 being a Na⁺-ATPase and the target of the spiroindolones. The physiological role of another putative Na⁺ transporter, the Na⁺/H⁺-exchanger PfNHE was also investigated, as previous studies on its contribution to regulation of [Na⁺]i and intracellular pH (pHi) have been controversial. On the basis of a bioinformatics analysis it was predicted that the protein functions as an amiloride-insensitive, plasma membrane Na⁺-extruder, like its closely related plant homologues. However physiological studies revealed no significant role for such an NHE in either pH<subscript>i or [Na⁺]i regulation in the P. falciparum trophozoite. This study constitutes a significant advance in our understanding of fundamental aspects of the cell physiology of the intraerythrocytic parasite, as well as shedding light on the mode of action of what promises to be an important new class of antimalarials, the spiroindolones.