Asymmetry-induced distinct mechanisms and the transporting role of sodium in bacterial fluoride channel Fluc

Bacterial fluoride channels (Fluc) are exceptional in their ability to conduct fluoride ions at rapid rates while maintaining extraordinary selectivity, an unusual property among anion transporters. Fluc achieves this through an antiparallel homodimeric assembly that forms two asymmetric ion-conduction pores and hosts a centrally bound sodium ion of previously uncertain function. Despite extensive structural characterization, the molecular basis for Flucs dual-pore asymmetry, transport efficiency, and sodium involvement has remained unresolved. Here, we performed long-timescale molecular dynamics simulations under electrophysiological conditions to elucidate the mechanisms of fluoride translocation through each pore. Our results reveal two distinct conduction modes: Pore I operates via the Channsporter mechanism, characterized by single-ion transport; while Pore II follows the Multi-ion mechanism in which paired fluoride ions exploit electrostatic repulsion to achieve faster sequential translocations. These findings demonstrate that structural asymmetry gives rise to mechanistic specialization within the same homodimeric protein. We further propose that the central sodium ion serves as a dynamic cofactor in fluoride transport, coupling its vertical oscillation to fluoride movement in a pore-dependent manner. Together, our results establish a unified model of asymmetric dual-pore conduction in Fluc and introduce a broader paradigm in membrane transport biology, one where inherent structural asymmetry enables divergent yet coexisting mechanisms of ion conduction. SignificanceFluoride is a naturally occurring environmental ion that is toxic to bacteria at elevated concentrations, necessitating specialized export systems for survival. Bacteria encode two distinct fluoride exporters: the CLCF antiporter and the Fluc channel. The latter of which remains mechanistically unresolved due to its unusual dual-pore architecture and lack of close homologs. Using extensive molecular dynamics simulations, we dissect fluoride permeation through Fluc and show that its two antiparallel pores operate through distinct transport mechanisms. Besides, we uncover a previously unrecognized role for the central sodium ion as an essential cofactor that enables fluoride conduction. Together, these findings provide new mechanistic insight into fluoride export and clarify the functional asymmetry underlying Fluc-mediated transport.