Malaria parasite type 4 equilibrative nucleoside transporters (ENT4) are purine transporters with distinct substrate specificity

Abstract

Malaria, caused by Plasmodia parasites, affects hundreds of millions of people. As purine auxotrophs, Plasmodia use transporters to import host purines for subsequent metabolism by the purine salvage pathway. Thus purine transporters are attractive drug targets. All sequenced Plasmodia genomes encode four ENTs (equilibrative nucleoside transporters). During the pathogenic intraerythrocytic stages, ENT1 is a major route of purine nucleoside/nucleobase transport. Another plasma membrane purine transporter exists because Plasmodium falciparum ENT1-knockout parasites survive at supraphysiological purine concentrations. The other three ENTs have not been characterized functionally. Codon-optimized Pf- (P. falciparum) and Pv- (Plasmodium vivax) ENT4 were expressed in Xenopus laevis oocytes and substrate transport was determined with radiolabelled substrates. ENT4 transported adenine and 2'-deoxyadenosine at the highest rate, with millimolar-range apparent affinity. ENT4-expressing oocytes did not accumulate hypoxanthine, a key purine salvage pathway substrate, or AMP. Micromolar concentrations of the plant hormone cytokinin compounds inhibited both PfENT4 and PvENT4. In contrast with PfENT1, ENT4 interacted with the immucillin compounds in the millimolar range and was inhibited by 10 μM dipyridamole. Thus ENT4 is a purine transporter with unique substrate and inhibitor specificity. Its role in parasite physiology remains uncertain, but is likely to be significant because of the strong conservation of ENT4 homologues in Plasmodia genomes.