Monovalent cations differentially regulate the activity of eukaryotic fluoride channels and impact fluoride resistance.

Fluoride is an environmentally prevalent inhibitor of fungi, plants, and other eukaryotes. The fluoride exporter (FEX) is the major resistance mechanism that prevents intracellular fluoride accumulation in eukaryotes. FEX activity is sodium-dependent, but the mechanism for Na+ activation and the impact of other cations on FEX function remain poorly understood. Here, we show that sodium and lithium have different effects on channel activity. We leverage these differences to understand how monovalent cations regulate FEX. Functional assays in a reconstituted system show that lithium acts as a competitive antagonist of channel activation by competing with sodium for the central cation binding site. We further demonstrate that lithium markedly reduces fluoride tolerance of yeast. A cryo-EM structure of Candida albicans FEX with Li+, together with molecular dynamics (MD) simulations, reveals that differences in the preferred coordination sphere underlie the cation dependence of FEX activity. Whereas sodium binding supports a dynamic structure with a broader pore radius, lithium coordination favors a more rigid conformation that is more compact in the channel's vestibule. These changes perturb the tilt angle of a pore lining helix, and alter the rotamer of a key phenylalanine in the pore, which together constrict the permeation pathway. In addition to providing general insight into the mechanism of fluoride channel regulation by monovalent cations, these results identify lithium as a previously unknown environmental antagonist of eukaryotic fluoride export and tie cellular fluoride stress tolerance to the abundance of additional ions in the cellular milieu.