A proton transfer mechanism in the malaria parasite lactate/H+ symporter reveals a channel-like transporter without conformational changes

The malaria parasite Plasmodium falciparum relies on anaerobic glycolysis for energy during the intraerythrocytic stage, producing lactate and H+ that must be extruded via its formate-nitrite transporter (PfFNT) to prevent death by cytosolic acidification and swelling. Unlike bacterial homologues classified as channels, PfFNT mediates saturable lactate transport and was classified as a transporter, raising questions about its transport mechanism and what distinguishes channels from transporters. Here, we combine over 720 s of molecular dynamics simulations, quantum chemical calculations, and transport assays in Xenopus laevis oocytes to characterize the transport cycle of PfFNT. We show a transport mechanism in which protonation of a central histidine (H230) enables lactate binding, and proton transfer from H230 to lactate is required to release the substrate as neutral lactic acid. This mechanism is supported by data indicating that neutral compounds such as lactamide do not enter the cavity, while anions like iodide, which are unlikely to protonate under physiological conditions, bind in the cavity but are not released and thus block lactate transport. As neither lactate nor lactic acid can diffuse passively through the protein, it is the requirement for proton transfer defines PfFNT as a lactate/H+ transporter rather than a channel. Notably, the PfFNT transport process occurs without significant protein conformational changes. Furthermore, our data indicates that under acidic conditions, small neutral molecules such as formic acid can passively diffuse through the protein. This suggests PfFNT challenges standard definitions of channels and transporters and differs from existing examples of channel-like transporters as it i) operates as a transporter without conformational changes and ii) can switch between transporter and channel modes within the same transport pathway.