The molecular mechanism of fluoride export by the eukaryotic fluoride channel FEX

Domain: neuroscience
Published: 2025-12-11
Editorial outcome: major_revision

Abstract
Much of life on Earth, including plants, fungi, and bacteria, evolved to resist toxic environmental fluoride. In eukaryotes, the major resistance mechanism is fluoride export by membrane proteins known as FEX. Using electrophysiology and transport assays, we establish that FEX from plants and yeasts are highly selective fluoride channels. Fluoride transport activity depends on reversible sodium ion binding, but sodium itself is not transported. We determine a structure of a FEX protein, from pathogenic yeast Candida albicans, using cryo-EM. Bolstered by mutagenesis studies, this structure reveals a fluoride permeation route through a single phenylalanine-lined pore. Molecular dynamics simulations demonstrate that a cation binding motif adjacent to the pore provides a stable sodium binding site that is accessible from the external aqueous solution. Comparison to the structurally related bacterial fluoride channels, Flucs, provides a glimpse of the evolution of structural and mechanistic complexity in a membrane protein family with inverted repeat architecture.

Introduction
Fluoride (F−) is present at biologically relevant concentrations throughout the natural environment, including soil, water, and certain minerals1. Human use of fluoride and fluorine-containing molecules has further compounded fluoride levels in some niches2. Intracellular fluoride accumulation inhibits multiple enzymes that are essential in multiple biological pathways, including glycolysis, ATP synthesis, phosphorylation, nucleotide synthesis, and other processes catalyzed by metalloenzymes3–5. As a result, most microorganisms, fungi, and plants possess mechanisms to avoid fluoride toxicity6,7. Understanding these fluoride resistance mechanisms is of interest to multiple fields, including biotechnology and synthetic biology, bioremediation, agriculture, and antimicrobial development2,8,9.

The major mechanism that plants and microorganisms use to protect against cytoplasmic fluoride accumulation is membrane export, and at least three types of membrane protein, including both antiporters and ion channels, have evolved to export this toxic halide10. Bacteria possess either F−/H+ antiporters from the CLC family of anion channels and transporters known as CLCF, or architecturally distinct fluoride channels known as Flucs11,12. In eukaryotes, including plants, fungi, and even some aquatic filter-feeding animals, fluoride export is managed by proteins known as FEX7,13,14. FEX posses the same fold as the Flucs, but are more architecturally complex. Whereas Flucs assemble as rare dual topology antiparallel homodimers, with one subunit inserted into the membrane facing out, and the other facing in ref.15, the FEX proteins have an inverted repeat architecture, in which their two homologous domains are synthesized as a single polypeptide with a transmembrane (TM) linker that enforces antiparallel assembly16. The antiparallel architecture of the Flucs represents an evolutionary antecedentoftheFEX17. Although inverted repeat architecture is extremely common among membrane transport proteins18, Fluc/FEX is the only family of known function in which both the antiparallel homodimer and fused inverted repeat versions are extant19. This provides an opportunity to understand how evolutionary forces shape membrane proteins after duplications and fusions of antiparallel domains, which occurred in the evolutionary history of a large proportion of membrane transporters18.

Functional, structural, and computational studies have yielded a detailed understanding of the architecture and fluoride transport mechanism of the Flucs. As a consequence of their antiparallel homodimeric architecture, the Flucs possess perfect two-fold symmetry20–22 and a pair of antiparallel, but otherwise identical pores22. Both pores simultaneously conduct fluoride23, which is expelled from the cell due to the cell’s resting negative membrane potential. Fluoride ions enter through Fluc’s large, electropositive aqueous vestibules, and are then translocated into the dehydrated part of the permeation pathway24,25. Here, the fluorides are coordinated by a track of polar residues, as well as the electropositive ring edges of a pair of phenylalanines from a motif known as the phenylalanine box22,26. The fluorides transit rapidly through this pore, with a rate of ~106 ions per second11. Anion selectivity occurs at two points along the pathway: at a conserved serine that translocates the second from the vestibule to the constricted part of the pore24,27, and, at the opposite end of the pathway, by a gate comprised of a threonine, glutamate, and tyrosine contributed by each of three different helices24,25. Both the vestibule serine and the gating triad residues are highly conserved in prokaryotes and eukaryotes. The Flucs also possess a sodium ion buried at the dimer interface. In structures, this sodium is bound by just four ligands, all of which are main chain carbonyls from the breaks in symmetry-related TM helices22. This sodium is not exchangeable on the day timescale in wildtype (WT) Fluc22, but has been shown to contribute to channel assembly28 and is required for activity29. The idiosyncratic architecture of the Flucs raises several questions about their structural relatives, the FEX. Is the symmetry and two-pore architecture of the Flucs preserved in the FEX? Are the channel mechanism and major chemical determinants of fluoride coordination retained? Does sodium or another cation play a role in the transport mechanism?

Prior functional studies, primarily with FEX homologs from Saccharomyces cerevisiae (S. cerevisiae) and Arabidopsis thaliana (A. thaliana), have helped narrow these knowledge gaps, but open questions remain. Whole-cell patch recording in yeast supported functional assignment of the FEX as ion channels16,30, but did not definitively rule out other mechanisms, for example, electrogenic proton-coupled transport, which has also been proposed31. Electrode-based efflux assays have confirmed strong selectivity against chloride, but transport of other halides or pseudohalides has not been assessed14. Sequence analysis and mutational studies suggest that the two domains of FEX are functionally asymmetric, with one functional and one vestigial pore13,16,22. However, even for the functional pore, some residues that are critical for Fluc activity cause only mild defects when mutated in FEX. For example, a key serine responsible for ion translocation from the vestibule to the pore in Fluc does not appear to be essential for FEX-mediated fluoride resistance13,24,27. The fluoride ion’s route through FEX thus remains uncertain. Finally, whether sodium plays a role in fluoride transport by FEX has not yet been explored.

Here we use planar lipid bilayer electrophysiology, liposome transport assays, yeast resistance assays, single-particle cryo-electron microscopy (cryo-EM), and computational simulations to establish the molecular basis of fluoride transport by FEX from plants and yeast. We find that these single-barreled electrodiffusive channels require sodium for activity, which binds reversibly to the WT channels through the external vestibule. A structure of FEX from the pathogenic yeast, Candida albicans (C. albicans) together with site directed mutagenesis, reveals a single narrow pore, lined by numerous phenylalanine residues, that bypasses the prominent vestibules characteristic of the Flucs. Together, these results provide molecular insight into fluoride transport and resistance in eukaryotes, and offer a glimpse into the evolutionary trajectory of a membrane protein from its most primitive dual topology form to complex inverted repeat architecture.

Results
Functional characterization of FEX homologs
We first surveyed genomes across the eukaryotic tree of life for FEX homologs, and found that most major eukaryotic clades include organisms that possess FEX-encoding genes (Fig. 1A). We screened multiple phylogenetically diverse FEX homologs for expression and biochemical behavior, achieving acceptable purifications of FEX proteins from plants A. thaliana (FEX-AT) and tea plant Camellia sinensis (FEX-CS), a globally important crop plant for which fluoride resistance is highly relevant32, and from yeasts C. albicans (FEX-CA) and S. cerevisiae (FEX-SC) (Supplementary Fig. 1). All of these FEX homologs catalyzed rapid fluoride efflux from proteoliposomes (Fig. 1B and Supplementary Fig. 2A), consistent with previous results14. Similar to the prokaryotic Fluc channels, the rate of fluoride transport by the plant FEX proteins exceeds the response time of the electrode, equivalent to a unitary turnover of >104 ions/s. In contrast, the yeast homologs exhibited slower transport kinetics, with rates of 2910 ± 110 s−1 for S. cerevisiae FEX and 1050 ± 90 s−1 for C. albicans FEX (Fig. 1B and Supplementary Fig. 3). We also screened additional halides and pseudohalides, including chloride, bromide, nitrate, cyanate, and thiocyanate, for transport by FEX-CA and FEX-CS using solid-supported membrane (SSM) electrophysiology, which detects transient currents elicited by electrogenic binding or ion transport into vesicles capacitively coupled to an electrode. In these experiments, only fluoride perfusion evoked a robust capacitive current. These experiments set a lower limit of 100-fold selectivity for fluoride over chloride, bromide, and nitrate for both FEX homologs. The fluoride-induced currents are at least 15-fold larger than those elicited by cyanate and thiocyanate. Because cyanate and thiocyanate generate a nonspecific signal even in protein-free samples, this likely represents an underestimate of FEX’s true selectivity against these pseudohalides (Supplementary Fig. 4).

The rapid fluoride transport by FEX-CS enabled planar bilayer electrophysiology with the purified proteins. For liposomes reconstituted at a low protein: lipid ratio, such that each liposome is expected to bear 1–2 FEX proteins based on a statistical analysis of liposome occupancy33, we observe fusion to the bilayer in extremely small, but discrete steps of ~0.5 pA at −200 mV under salt gradient conditions (Fig. 1C). Like Fluc-Bpe, the single channel currents do not exhibit sub-states or closures, and are fully open at potentials from −200 to +200 mV. A histogram of conductances measured for single insertion events obtained from different bilayers exhibits a major population with a mean of 2.2 pS, and a second, smaller population centered at a value approximately twice as large (4.5 pS). These peaks most likely represent the fusion of singly- and doubly-occupied FEX-CS liposomes, yielding a mean single channel conductance of 2.2 pS. This rate of fluoride permeation is >10-fold faster than expected for a transport mechanism that requires a conformational change in the protein.

After several dozen channel fusions, we measured the current–voltage (I–V) relationship for FEX-CS under three different fluoride gradients (Fig. 1D, E). The measured reversal potentials were Nernstian with respect to the fluoride activity gradient (Fig. 1F). This experiment provides unequivocal evidence that fluoride permeation is not coupled with any other ion. FEX-AT behaved similarly to FEX-CS, with a 2.5 pS mean conductance and reversal potentials consistent with channel activity under fluoride gradient conditions (Supplementary Fig. 2B–D). These experiments together confirm that eukaryotic FEX acts via a channel mechanism, with substantial differences in the ion transport rates among different organisms.

Cryo-EM structure of FEX from Candida albicans
We next sought to determine the structure of a FEX homolog using cryo-EM. At just ~40 kDa, and without any extra membrane domains, FEX channels are a challenging target for cryo-EM analysis. To increase the particle size and aid particle alignment, we appended the 17-residue membrane-proximal external region (MPER) epitope as a continuous extension of the N-terminal TM helix of FEX-CA. This epitope, derived from the envelope glycoprotein gp41 in human immunodeficiency virus (HIV), has been shown to provide a rigid, membrane-adjacent docking site for several well-characterized monoclonal antigen-binding (Fab) fragments34–37 that can serve as cryo-EM fiducials for membrane proteins during single particle cryo-EM analysis38,39. In addition, the first 75 residues of FEX-CA, which are not conserved and are predicted to be disordered, were truncated to decrease structural heterogeneity. This engineered FEX-CA, with the MPER epitope, is biochemically stable and maintains comparable activity to the full-length FEX-CA (Supplementary Figs. 1 and 5A). The Fab 10E8v4 forms a stable complex with MPER-tagged FEX that is monodisperse by size-exclusion chromatography (SEC) (Supplementary Fig. 5B) with homogenous particles (Supplementary Fig. 6A). Binding of this Fab has no effect on the fluoride efflux activity of FEX-CA (Supplementary Fig. 5A).

The Fab-bound MPER-FEX-CA, solubilized in DDM micelles, was used for single particle cryo-EM analysis. Selected 2D averages of Fab-bound proteins in different orientations exhibit TM domain features (Supplementary Fig. 6A). After multiple rounds of ab-initio 3D reconstruction, 3D classification, heterogenous refinement, and non-uniform refinement, we obtained a 4.0 Å density map of MPER-FEX-CA bound to the 10E8v4 Fab, with local resolution up to 3.5 Å in the protein core (Fig. 2A; Supplementary Figs. 6; 7 and Supplementary Table 1). In this map, the densities for the constant and variable domains of the 10E8v4 Fab are well resolved, and in FEX-CA, all nine TM α-helices and their connecting loops have clearly resolved side chains sufficient for model building and residue assignment (Supplementary Fig. 8). Docking the known 10E8v4 structure (PDB 5IQ9)37 into the map shows that the Fab fragment’s interactions with FEX-CA are almost entirely restricted to the MPER epitope, avoiding perturbations of mechanistically important parts of the protein, a common problem for fiducials selected from randomized libraries40. We built an atomic model for residues 1–381 of the engineered FEX-CA construct, corresponding to 76–381 of WT FEX-CA (Supplementary Table 1). Only nine residues from the unstructured C-terminus were missing from the density map and could not be modeled. This structure shows that FEX-CA has homologous N-terminal (TM1–4) and C-terminal domains (TM6–9), which comprise the protein core. The domains are connected by a transmembrane linker helix (TM5) that maintains the antiparallel domain architecture (Fig. 2B). As in the Flucs, the third helix in each domain (TM3 and TM8) is broken by a stretch of approximately three non-helical residues. These breaks in the α-helices crossover each other at approximately the midpoint of the membrane. Compared to our experimental structure, the model predicted by AlphaFold 341 has a root mean squared deviation (RMSD) of 2.5 Å for 237 Cα atoms within the TM helices. While the protein core (N- and C-terminal domains) of the experimental and Alphafold models are similar (RMSD of 1.6 Å for 213 Cα atoms in the TM helices), significant deviations occur in the vicinity of TM5, the linker helix (Supplementary Fig. 9).

Architectural asymmetry of FEX
FEX possesses internal C2 pseudosymmetry with the N- and C-terminal domains tilted along a symmetry axis tilted approximately 10° relative to the plane of the membrane (Fig. 2C). This contrasts with the Flucs, which exhibit perfect 2-fold domain symmetry about an axis parallel to the plane of the membrane22,40. The N- and C-terminal domains of FEX-CA align well across TM α-helices 1–3, with an RMSD of 2.5 Å. However, rigid body rotations of fourth helix in each domain, TM4 and TM9, in relation to the rest of their domain bundle introduces a ~20° offset in the structural alignment of the N- and C-terminal domains (Supplementary Fig. 10). A nonprotein density consistent with a lipid or a detergent molecule segregates the linker helix, TM5, from the protein core, so that the core and linker are limited to a single point of contact: one putative hydrogen bond between S235 in the linker helix and H80 in the N-terminal core domain (Fig. 2C and Supplementary Fig. 11). These structural observations are consistent with the lack of conservation in TM5 sequences among eukaryotic FEX.

In the Fluc dimers, the crossover of the broken helices 3 and 3′ defines the channels’ most prominent feature, a pair of aqueous, electropositive vestibules. These vestibules serve as an entry point and electrostatic funnel for second before they enter the constricted part of the pore24,25. The vestibule architecture is substantially different in FEX-CA. The tilt of the homologous domains effectively broadens the external vestibule and narrows the cytoplasmic vestibule. Moreover, the cytoplasmic vestibule of FEX-CA is plugged by the loop between TM2 and TM3a, which forms a short helical segment and extends the bulky side chain W127 into the cleft defined by the broken helices of TM3 and TM8 (Fig. 2D). Because of this occlusion, there is little aqueous volume in the cytoplasmic vestibule. Meanwhile, the external vestibule, while water-filled and of similar size as the vestibules in the Flucs, is strikingly electronegative. Several acidic side chains, including E165, E333, D336, and D337, line the aqueous surface (Fig. 2D). These electrostatics would disfavor fluoride entry into the external vestibule, which is likely to be adaptive in the presence of high environmental fluoride.

In lieu of a commodious vestibule to funnel fluoride ions into the pore, our FEX-CA model instead reveals a long, narrow crevice surrounded by TM3b, TM4, TM8b, and TM9 (Fig. 2D). This route traverses almost the entire length of the protein, interrupted only by the conserved triad of T293, E352, and Y364. In Flucs, this triad contributes to ion selectivity24; the position of these interacting residues on the cytoplasmic side of the pore in FEX is consistent with a role as the primary selectivity filter for fluoride ion.

The fluoride permeation pathway in FEX-CA partially coincides with one of the pores of the double-barreled Flucs, skirting the two conserved edges of the phenylalanine box motif (F159 and F349) and the conserved N297 near the mid-point (Fig. 2D and Supplementary Fig. 12). Consistent with mutagenic studies13, this structure shows in FEX, the second pore that characterizes the Fluc channels has degraded. The opposite two edges of the phenylalanine box motif have been replaced by two valine residues, and the vestigial route is tightly packed with hydrophobic side chains (Fig. 2D).

The fluoride permeation pathway is lined by phenylalanines and bypasses the vestibules
The crevice is lined by several polar residues expected to be involved in fluoride permeation, including residues T293, E352, and Y364 from the selectivity triad, and the polar track side chain N297 (Fig. 3A). These positions are highly conserved from bacteria to eukaryotes, and have been shown to be essential for fluoride resistance by S. cerevisiae FEX13,16. In addition to these expected polar sidechains, the FEX-CA pore is also lined by residues that have not previously been implicated in eukaryotic fluoride resistance, notably a confluence of phenylalanines surrounding the pore along its entire length (Fig. 3A). The conserved phenylalanines from the phenylalanine box, F159 and F349, which have previously been shown to contribute to FEX function13 are joined by F167, F191, F373, and F294 to line the putative permeation route. Coordination by the electropositive quadrupolar edge of aromatic side chains has emerged as a theme in biological fluoride recognition, so it seems likely that these phenylalanines also contribute to fluoride permeation22,26,42.

To test this, we introduced constructs with phenylalanine-to-isoleucine point mutations into a fluoride-sensitive yeast strain (S. cerevisiae Δfex1 Δfex27) and monitored the fluoride resistance relative to cells expressing WT FEX-CA (Fig. 3B). We selected isoleucine sidechains to maintain the hydrophobicity and size of phenylalanine, while eliminating the polarizability of the aromatic ring. Although Western blot analysis showed that the membrane expression varied as much as five-fold among these constructs (Supplementary Fig. 13B, C), the expression level was not a decisive factor in the fluoride resistance phenotype. For example, F349I exhibited the lowest expression level among the mutants, at just 20% of WT, and yet showed the most robust fluoride resistance among the mutant constructs. F349I thus provides a benchmark for a lower limit of FEX expression that still rescues the fluoride-sensitive strain.

These experiments support the hypothesis that the Phe box residues contribute to fluoride resistance: the F159I mutant did not grow at 0.5 mM NaF, whereas F349I was less impaired, only showing sensitivity at 5 mM NaF that might be explained by this construct’s lower expression level (Fig. 3B and Supplementary Fig. 13A). This is consistent with previous observations in bacteria and yeast, where mutation of the F349 equivalent is less detrimental than mutation of the F159 equivalent13,22. Mutations to the other Phe residues that line the permeation pathway, F167I, F191I, F373I, and F294I, all cause substantial fluoride sensitivity at 0.5 mM NaF (Fig. 3B), establishing the pore-lining phenylalanines as a key component of fluoride export activity in vivo.

Notably, the pore crevice bypasses the cytoplasmic aqueous vestibule, which is functionally important in the Flucs. However, it is conceivable that rearrangement of the TM2–TM3 loop segment could unplug FEX-CA’s vestibule and present an alternative entry point for fluoride into the pore. We therefore sought further functional confirmation that the cytoplasmic vestibule is not part of the fluoride permeation route. To test this, we mutated S347 (Fig. 3A), a position analogous to the vestibule serine in the Flucs that is proposed to rotamerize to deliver fluoride from the vestibule to the pore24,27. In the Flucs, mutation to the smaller residue alanine has little impact on fluoride transport, but introducing the conformationally restricted threonine at this position abolishes activity24. In contrast, in FEX, the analogous mutation S347T shows robust rescue of the fluoride-sensitive phenotype (Fig. 3B and Supplementary Fig. 13A). This suggests that a similar conformational change is not required of the vestibule-facing S347, and, by inference, that ion translocation from the cytoplasmic vestibule to the pore is not a feature of FEX’s transport mechanism. Other mutations introduced at this position in the S. cerevisiae homolog have similarly been shown to be functionally mild13,16. Meanwhile, in the FEX-CA structure, the symmetry-related serine, S160, is rotated away from the external vestibule, in an orientation similar to the alternate rotamer proposed, but not structurally verified in the Flucs24,27. The FEX-CA density map shows that the S160 sidechain interacts with the T301 sidechain from TM7, ~5 Å away from the pore crevice (Supplementary Fig. 14). In contrast to S. cerevisiae FEX, which is relatively insensitive to mutation at this position16, in C. albicans FEX, S160A and S160T both greatly diminish fluoride resistance (Fig. 3B). However, given this sidechain’s distance from both the vestibule and the crevice and its lack of functional conservation in the S. cerevisiae protein, we think it is unlikely that S160 is directly involved in fluoride transport by FEX.

Sodium is essential for FEX channel activity
In the Flucs, structures show a sodium ion is buried at the dimer interface, coordinated by four carbonyl oxygen atoms from the crossover region of TM3 helical breaks22. The sodium is inserted during Fluc assembly and is not exchangeable in WT Fluc28,29. It is not known whether Na+ or other cations play any role in FEX structure or function.

To investigate whether Na+ contributes to FEX activity, we examined fluoride efflux from proteoliposomes as a function of Na+ concentration for WT FEX-CA and FEX-CS. For both homologs, we observe a sodium dose-dependent increase in total fluoride efflux (Fig. 4A), reflecting a change in the fraction of liposomes that possess an active fluoride exporter. Western blot analysis of the proteoliposomes confirmed that this trend is not related to protein reconstitution efficiency (Supplementary Fig. 15). We instead interpret this result to mean that the relative proportion of active and inactive FEX varies as a function of Na+ concentration. Using Poisson distribution statistics to correct for liposomes that contain multiple channels33, we converted our measurement of transport-active liposomes into an estimate of the fraction of active protein at each Na+ concentration. This relationship is well fit by a single-site binding isotherm for both FEX-CA and FEX-CS, with apparent dissociation constant (Kd) values of 0.4 and 0.5 mM, respectively (Fig. 4B). It is notable that for both homologs, there is residual fractional activity in the absence of Na+, which is unexpected for an equilibrium two-state model if Na+ is the only cation that binds FEX and supports fluoride efflux. We therefore considered that K+ might substitute for Na+ at the high concentrations (300 mM K+) used for these assays. Indeed, when the background K+ concentration is reduced to 100 mM, the proportion of transport-inactive liposomes increases (Supplementary Fig. 16), consistent with weak K+ binding supporting fluoride efflux. Based on the residual fractional activity in the absence of Na+, we estimate that FEX-CA and FEX-CS bind K+ with ~500 and ~650 mM affinity, respectively. Assuming that these cations compete for a single site, we estimate that the Kd for Na+ in the absence of K+ is ~0.2 mM for FEX-CA and ~0.3 mM for FEX-CS, and thus that the site favors Na+ over K+ by over three orders of magnitude. Li+, in contrast, inhibits fluoride export activity by FEX-CA and FEX-CS, with fluoride export reduced to the level of protein-free liposomes at 10 mM Li+ (Fig. 4C).

To approximate the kinetics of cation exchange, we exploited the differential effects of Li+ and Na+ on fluoride efflux activity to assess Li+/Na+ exchange. We reconstituted FEX-CA proteoliposomes with 0.6 mM Li+. We then added 10 mM Na+ to the experimental buffer and monitored the fractional activity of the sample as a function of time. In these experiments, fluoride efflux increases over time, corresponding to substitution of the initial Li+, which suppresses activity, with Na+, which supports activity (Fig. 4D). This experiment provides evidence of reversible cation binding to the protein in lipid membranes. Although it is not possible to tease apart individual rate constants in this experiment, the t1/2 for exchange of 2 h suggests that cation association and dissociation occur on a tens-of-minutes-to-hour timescale.

While we do not see sodium in our map, and do not expect to at this resolution, the protein is structured to accommodate a sodium at a similar position as the Flucs. Well-defined density of the crossover region shows four carbonyl oxygens made available by the TM breaks poised near the bottom of the external vestibule, arranged in a cation-binding motif called a niche43 (Fig. 4E). These positions, G154, S157, G341, and T344, are equivalent to those that bind Na+ in the Flucs. In our structures, the niche motif appears to be at least partly exposed to the aqueous solution from the external vestibule and inaccessible from the cytoplasmic side. The pore-lining residues F159 and F349 are immediately adjacent to the residues that comprise the niche motif, and pore-lining N297 also hydrogen bonds with the helical breaks, potentially linking cation binding and fluoride export activity.

To evaluate the stability of sodium and potassium in this putative binding site, we performed molecular dynamics simulations of FEX-CA with a Na+ or K+ initially placed in the niche. In each of three approximately 750 ns simulations, the Na+ ion remained coordinated by the four carbonyl oxygens atoms at close distances for the entire simulation (Fig. 4F; Supplementary Figs. 17 and 18A), in agreement with the slow kinetics of the experimentally measured cation exchange. Similar behavior was also observed for the Na+ ion when the system was prepared with NaF rather than NaCl (Supplementary Fig. 18). The stable coordination of the Na+ is supported by a strong electrostatic attraction, with a van der Waals (vdW) component consistent with a close interaction between the Na+ and FEX residues (Supplementary Fig. 18B). The residues that coordinate the Na+ ion exhibit a low root mean squared fluctuation (RMSF) relative to the rest of the protein (Supplementary Fig. 18C).

In contrast, when simulations were performed with the weakly interacting K+, the cation was held more loosely and tended to move out from the binding motif. The average RMSD of FEX is similar in the presence of both cations, but the RMSD for K+ is larger than that of Na+, reflecting greater displacement of K+ (Fig. 4F). Figure 4G tracks the cations’ movement along the Z- and X-axes, and illustrates movement of K+ toward the external vestibule.

In these simulations, water penetrates the protein, especially the external vestibule (Fig. 4H). Water molecules from the external vestibule move in and out of the cation’s coordination spheres, typically exchanging quickly with other nearby waters (Fig. 4I, J). Compared to Na⁺, K⁺ is surrounded by more water (Fig. 4K), which could contribute to its displacement from the niche.

The electrostatic interaction energies indicate a favorable interaction between the Na⁺ ion and nearby water molecules. Initially, the electrostatic energy is close to zero when no water molecules are present around the sodium ion, but becomes increasingly negative as hydration occurs during the simulation (Supplementary Fig. 18D). Most commonly, a single water coordinates Na+ from an axial position, yielding distorted trigonal bipyramid sodium coordination geometry, although instances of two waters binding were also observed, yielding 6-coordinate distorted octahedral geometry (Fig. 4K). Thus, these simulations demonstrate that the cation niche provides a binding site for sodium ion that is accessible to the external vestibule, complementing our experimental observation of cation exchange.

Methods
Expression constructs and mutagenesis
Protein expression constructs (Supplementary Table 2) were composed of codon-optimized synthetic genes encoding FEX homologs (Integrated DNA Technologies, Coralville, IA) with the MPER tag38, an N-terminal pre-signaling peptide of α-mating factor to improve mature protein secretion and insertion into the S. cerevisiae plasma membrane52, and a C-terminal streptavidin tag for protein purification. To predict the best site of fusion for the MPER epitope tag, we prepared a homology model of the N-terminal FEX domain based on the Fluc-Ec2 structure and appended the tag at the position that corresponded to previously determined structures of MPER-Fluc-Ec2 in complex with antibody38. We assessed two antibody fragments, 10E8v4 and VRC42, for binding to this MPER-FEX-CA construct, and selected 10E8v4 based on the monodispersity of the complex in size exclusion chromatography. These constructs were cloned into the pDD_Leu2D vector53 and transformed using a standard lithium acetate method into S. cerevisiae strain BJ2168 for protein expression, or Δfex1 Δfex2 SSY37 for yeast resistance assays. All constructs were verified by sequencing. Mutagenesis was performed using overlap extension PCR with primers synthesized by Integrated DNA Technologies (Supplementary Table 3).

FEX expression, purification, and reconstitution
Starter cultures were prepared from yeast transformants in leucine dropout media (SC-Leu, Sunrise Science Products, Knoxville, TN) with 2% glucose, and incubated overnight at 30°C, 240 rpm prior to inoculation of protein expression cultures in SC-Leu with 2% lactate and 0.2% glucose. These were grown (30°C, 240 rpm) until an OD600 of 0.8, then induced with galactose (final concentration 2% v/v). After 24 h, cell pellets were harvested, washed with Tris-EDTA (TE) buffer, snap-frozen in liquid nitrogen, and stored at −80°C until use.

Protoplasts were prepared from yeast pellets using Zymolase 20T digestion of the cell wall (20 U/g cell pellet) according to the manufacturer’s protocol (Amsbio, Cambridge, MA). Protoplasts were harvested by centrifugation, resuspended in breaking buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM NaF), and lysed using ~25 strokes in a pre-chilled Dounce homogenizer. Lysate was extracted with 2% n-Dodecyl-β-D-Maltoside (DDM) (Anatrace, Maumee, OH) for 2 h at room temperature. After pelleting cell debris, supernatant was applied to a gravity column with Strep-Tactin XT 4Flow resin (IBA Lifescience, Pittsburgh, PA) equilibrated with wash buffer (breaking buffer with 1 mM DDM). Protein-loaded resin was washed with 20 column volumes (CV) of wash buffer and eluted with 6 CV of wash buffer with 50 mM biotin. Eluate was further purified by SEC (Superdex 200, Cytiva, Marlborough, MA) in SEC buffer (20 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) pH 7.5, 150 mM NaCl, 5 mM NaF, and 0.5 mM DDM for cryo-EM or 1 mM DDM for functional studies). Fluc-Bpe was expressed and purified exactly as previously described22.

Freshly purified protein was added to Escherichia coli (E. coli) polar lipids (EPL; Avanti Polar Lipids, Alabaster, AL) solubilized with 35 mM 3-[(3-Cholamidopropyl)-Dimethylammonio]-1-Propane Sulfonate (CHAPS; Anatrace, Maumee, OH), followed by dialysis against 4–6 L of reconstitution buffer (typically 15 mM HEPES pH 7.5, 300 mM KF and 10 mM NaF) over 1.5 days. For cation-dependent experiments, NaF was substituted with NaCl, N-methyl-glucamine chloride, or LiCl as indicated. After dialysis, protein/lipid mixtures were subjected to three freeze/thaw cycles and extruded through a 400 nm filter (Cytiva, Marlborough, MA) to generate unilamellar proteoliposomes. Proteins were reconstituted at a protein: lipid ratio of 1 µg protein/mg EPL (protein:lipid molar ratio of ~1:50000) for the FEX homologs or 0.2 µg protein/mg EPL (molar ratio of 1:200000) for Fluc-Bpe.

Fab preparation
10E8v4 and VRC42 antibody expression vectors were obtained from the AIDS Reagent Program, Division of AIDS, NIAID, NIH, from Dr. Peter Kwong37,38,54. Heavy and light chain DNA was transfected into ExpiCHO-S and expressed using ExpiFectamine CHO reagents (Thermo Fisher Scientific) according to the manufacturer’s protocol. Six days post-transfection, supernatant was collected and the IgG-10E8v4 purified by gravity-flow affinity chromatography using protein A Sepharose 4B resin (Invitrogen) and eluted with Pierce IgG Elution buffer (Thermo Fisher Scientific). After pH neutralization, the 10E8v4 Fab fragment was generated by papain cleavage (Sigma-Aldrich 1:200 v/v) in phosphate-buffered saline with 1 mM EDTA and 6.5 mM cysteine, followed by protein A affinity column purification to separate the Fc domain and Fab fragment, and SEC (Superdex 200, 20 mM HEPES pH 7.5, 150 mM NaCl, and 5 mM NaF).

Cryo-EM grid preparation and data collection
MPER-tagged FEX-CA was incubated with the 10E8v4 Fab at a molar ratio of 1:1 for 1–2 h at room temperature. The complex was isolated using a Superdex 200 column equilibrated in SEC buffer. This sample was concentrated to 3–4 mg/mL, and 3.5 µl was applied to glow-discharged copper Quantifoil R1.2/1.3 300 mesh grids (Electron Microscopy Sciences, Hatfield, PA, USA). Grids were blotted for 2.5 s at 18 °C, 100% humidity and plunge-frozen in liquid nitrogen cooled liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). Grids were imaged on a Titan Krios G3 electron microscope (Thermo Fisher Scientific) operated at 300 kV and equipped with Gatan K3 direct electron detector and a Bioquantum Imaging Filter (slit width 20 eV). Micrographs were collected at magnification of ×81,000 (1.1 Å per pixel) or ×105,000 (0.834 Å per pixel) with a defocus range from −0.8 to −2.5 µm using SerialEM v4.1.155. Each image stack was composed of 50 individual frames with a total dose of 50 e− per Å2 (for ×105,000 magnification) or 60 e− per Å2 (for ×81,000 magnification).

Cryo-EM image processing
The cryo-EM workflow is shown in Supplementary Fig. 7. An initial template for particle picking was generated from 4702 images collected at a magnification of ×81,000 (Supplementary Fig. 7A). After patch-motion correction and patch contrast transfer function (CTF) estimation in cryoSPARC v4.256,57, particles were picked using crYOLO v1.8.458, extracted with a box size of 256 pixels (1.1 Å/pixel), and binned to 64 pixels in cryoSPARC v4.2. After several rounds of 2D classification and ab initio reconstruction (5 classes), a volume representing the FEX-CA/10E8v4 complex was selected as a template for automatic particle picking for 21,384 patch-motion corrected and CTF-corrected images combined from 3 datasets (×81,000). After many rounds of iterative 2D classification with extracted particles, 938,364 particles were selected and subjected to four rounds of heterogeneous refinement with the previously generated templates, followed by a 2-class ab initio reconstruction. After ab initio reconstruction, 142,174 particles were then subjected to a subsequent round of 3D classification with a solvent mask, and 54,516 particles were selected and refined to 7.28 Å resolution using non-uniform refinement. This map showed clear density for the TM helices, but the resolution was insufficient to build an atomic model. We used this low-resolution map to generate 3D template volumes for a FEX-CA/Fab complex, Fab alone, FEX-CA with no Fab, and empty micelles. These four 3D volumes were then used as templates for isolating particles for high-magnification datasets (Supplementary Fig. 7A—blue box).

An additional 41,056 images were collected at a magnification of ×105,000 and pre-processed using patch-motion correction and patch CTF correction in cryoSPARC v4.5.1 (Supplementary Fig. 7B). After micrograph denoising, 36,508 images were subjected to templating picking using the low-resolution map of the FEX-CA/Fab complex. Particles were extracted with a box size of 300 pixels (0.834 Å/pixel), followed by heterogeneous refinement with the four 3D template volumes generated earlier (Supplementary Fig. 7A—blue box). The class representing the Fab/FEX complex was subjected to a 4-class ab initio reconstruction followed by heterogeneous refinement. After 3–4 rounds of 2-class ab initio reconstruction followed by heterogenous refinement, a maximal number of complex particles had been obtained. These 439,352 particles were further processed using 2-class ab initio reconstruction followed by 4 rounds of heterogeneous refinement, yielding a well-resolved 4.14 Å map of the complex with 280,006 particles. To further improve the map resolution, these particles were re-extracted with a larger box size of 352 pixels (0.834 Å/pixel) for iterative 2D classification, 2-class ab initio reconstruction, heterogeneous refinement, and nonuniform refinement, yielding a final 4.0 Å map derived from 164,326 particles (Supplementary Fig. 7C).

Model building, refinement, and structural validation
The 10E8v4 Fab structure (PDB: 5IQ9)37 and the FEX-CA Alphafold259 prediction were used as initial models to fit into the experimental maps followed by manual adjustment in Coot (v0.9.8.93)60 and real-space refinement in PHENIX (v1.21)61. Structural validation was performed using PHENIX and MolProbity62. PyMOL v3.1 (Schrödinger) and ChimeraX v1.863 were used to visualize the structural data and generate figures.

Fluoride efflux assay
Fluoride efflux from proteoliposomes was measured using a lanthanum fluoride electrode (Cole-Parmer, Vernon Hills, IL) as previously described in detail11,64. Briefly, proteoliposomes were passed over Sephadex G50 resin equilibrated in assay buffer (typically 300 mM K+ isethionate, 1 mM KF, 10 mM HEPES-KOH, pH 7.5) and diluted 40-fold in assay buffer. Fluoride efflux was initiated by the addition of valinomycin (Sigma-Aldrich) to a final concentration of 1.8 µM. At the end of the experiment, proteoliposomes were disrupted by the addition of 30 mM n-octyl-β-D-glucoside (β-OG) (Anatrace, Maumee, OH) to release remaining encapsulated fluoride. To convert the fraction of occupied liposomes to the fraction of active protein Af, a correction was applied assuming that proteins were inserted into liposomes according to Poisson statistics33:

Af = k ln(f0)

where f0 represents the fraction of liposomes that do not exhibit protein-dependent efflux, and k is a constant that represents the maximum amount of protein-dependent efflux, which we determine empirically at saturating Na+.

Solid-supported membrane (SSM) electrophysiology
Experiments were performed using a SURFE2R N1 instrument (Nanion Technologies, Munich, Germany). 3 mm-diameter sensors were prepared with a 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) lipid monolayer as previously described65. FEX-CA and FEX-CS proteoliposomes (10 µg protein/mg EPL) were diluted 5-fold in assay buffer (10 mM HEPES, pH 7.5, 100 mM KCl, 5 mM Na+ isethionate) and absorbed to the DPhPC lipid monolayer. Potassium salts of each test anions was prepared at 10 mM in assay buffer. A full experiment protocol included a 2 s perfusion with assay buffer, followed by 2 s perfusion with test anion, and a final 2 s perfusion with assay buffer to return to the equilibrium condition. Each anion series was tested in triplicate using a single sensor.

Planar lipid bilayer electrophysiology
Planar lipid bilayer electrophysiology was performed using a horizontal planar lipid bilayer system separated by a partition with cis side (upper chamber) and trans side (lower chamber, electrical ground)11,66. For single-channel recording, FEX-CS or FEX-AT proteoliposomes are fused with a 3:1 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)/1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) (Avanti Polar Lipids, Alabaster, AL) bilayer, with 300 mM NaF, 15 mM 3-(N-morpholino)propanesulfonic (MOPS) pH 7 in the cis chamber, and 30–300 mM NaF, 15 mM MOPS pH 7 in the trans chamber, respectively. Current output from an Axopatch 200B amplifier was recorded using Clampex and electronically low-pass filtered at 1 kHz and digitally filtered at 50 Hz for data analysis in Clampfit 10.6 (Molecular Devices, San Jose, CA).

Fluoride resistance in yeast
The Δfex1 Δfex2 yeast strain SSY37 was transformed with FEX-CA mutant plasmids using a standard lithium acetate protocol. Transformants were selected on plates prepared with uracil dropout medium (SC-Ura, Sunrise Science Products, Knoxville, TN) with 2% glucose. For yeast resistance assays, overnight cultures were diluted to an OD600 of 1 and 10-fold serial dilutions were spotted on SC-Ura plates with 2% galactose to induce FEX expression and NaF as indicated. Plates were imaged after 2–3 days at 30°C. Expression and membrane localization were validated by Western blot. Cells were collected after 24 h galactose induction and lysed using Zymolase treatment followed by vortexing. After removing cell debris, the membrane fraction was isolated by ultracentrifugation and membrane pellets were resuspended in buffer containing 4% SDS and 4 M urea. MPER-tagged FEX was detected using purified VRC42 IgG primary antibody54 in a 1:2500 dilution. As a loading control, the constitutively expressed plasma membrane H+-ATPase (Pma1) was detected using an anti-Pma1 primary antibody (Thermo Fisher Scientific Cat# MA1-91567, RRID: AB_1959827) in a 1:1000-fold dilution.

Molecular dynamics simulations and analysis
The starting structure of FEX-CA was generated using the current cryo-EM structure (PDB: 9OP1). This structure was aligned with Fluc-Bpe (PDB: 5NKQ) to place the Na+ or K+ ion in the niche. Afterwards, the protonation states of all titratable residues at pH 7.4 were determined using the H++ web server67,68. The protein was inserted into a 3:1 POPE/POPG bilayer and solvated with TIP3P69 water molecules using Packmol-Memgen70. Missing hydrogen atoms and Na+ ions were added to neutralize the system, and NaCl, NaF, or KCl ions were then added using the tleap module from the AmberTools2071 to achieve a physiological salt concentration of 0.150 M. All systems consisted of 98,092 atoms within a solvation box of 105 Å × 105 Å × 104 Å, containing approximately 17,758 water molecules. The initial coordinate and parameter files were generated using tleap71, applying the Amber ff14SB72, lipid2173, and TIP3P69 force fields.

Three independent simulation trials were performed using the pmemd.cuda module of the Amber 20 software package70,74 for each of the systems solvated in NaCl, NaF, or KCl to ensure adequate sampling. To properly relax and minimize the system, we used a multistep energy minimization protocol in which each step involved both the steepest descent and conjugate gradient methods. Specifically, the first two steps consisted of 5000 iterations with steepest descent followed by 5000 iterations with conjugate gradient. In the final step, each method was applied for 10,000 iterations. Initially, only water molecules and hydrogen atoms were minimized, while a positional restraint of 7.5 kcal/mol/Å2 was applied to all other atoms. In the second stage, minimization was extended to include ions and protein side chains, with the restraint on the protein backbone. In the final step, all restraints were removed, allowing the entire system to relax freely.

Following minimization, the system was heated in two steps. First, the temperature was gradually increased from 0 to 100 K over 4 ns using the Langevin thermostat75, with restraints of 5 kcal/mol/Å2 applied to the protein and lipids. In the second step, the temperature was raised from 100 to 303 K over 8 ns. During this stage, the Berendsen weak-coupling barostat76 was used alongside the Langevin thermostat to equilibrate the pressure, while maintaining the same positional restraints. The system was then equilibrated through 10 sequential steps, each lasting 0.5 ns, to stabilize the periodic boundary conditions. Following equilibration, 750 ns of production simulations were performed for each trial. A 2 fs timestep was used, and all bonds involving hydrogen atoms were constrained using the SHAKE algorithm77. A 12 Å cutoff was applied for long-range non-bonded interactions. Simulations were conducted in the NPT ensemble, with the temperature maintained at 303 K using the Langevin thermostat76 and pressure controlled by the Berendsen barostat76. The production frames were saved every 10 ps. Simulation input and parameter files for all the systems are available at Zenodo [https://doi.org/10.5281/zenodo.17343824]. It is important to note that accurate atomistic modeling of F− permeation or binding is intrinsically challenging due to its high charge density and strong hydration, which might not be fully captured by standard nonpolarizable force fields. Accordingly, our simulations were not designed to resolve detailed fluoride transport energetics or permeation pathways.

All analyses were performed using the cpptraj module from the AmberTools2078 and VMD software79 on every 100 ps of all production trajectories. Root mean squared displacement (RMSD) and RMSF were calculated to evaluate the structural stability. The number of water molecules within 3 Å and 7 Å of the Na+/K+ ion was counted, corresponding to its first and extended coordination shells80. Electrostatic and vdW interaction energies between the cation-binding motifs and the Na+/K+ ion, between the Na+/K+ ion and all water molecules within 3 Å, were computed using linear integration energy (lie) analysis. Visualization of the simulations and structural data, as well as figure generation, was performed using VMD software79.

AlphaFold models
Models of FEX proteins from Trypanosoma cruzi (FEX-TC), Noctiluca scintillans (FEX-NS), Tribonema minus (FEX-TM), Chlorella vulgaris (FEX-CV), Oryza barthii (FEX-OB), Camellia sinensis (FEX-CS), A. thaliana (FEX-AT), Musa balbisiana (FEX-MB), Ciona intestinalis (FEX-CI), S. cerevisiae (FEX-SC), Cladophialophora carrionii (FEX-CC), and Aspergillus niger (FEX-AN) were generated using AlphaFold341. Models overlaid with pLDDT scores (two views) and predicted aligned error plots are shown in Supplementary Fig. 21, along with Uniprot identifiers for each protein sequence. Source data (protein sequences used to generate models) are provided as a source data file.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript by the Stockbridge and Ohi labs describes the structural basis of fluoride transport by the FEX channels. These widespread channels mediate fluoride exports in many unicellular eukaryotes and plants, to allow these organisms to overcome environmental fluoride toxicity. The lab had described the prokaryotic fluoride channels Flucs, and shown they are dual topology proteins. Here, the authors show that the FEX proteins are also ion channels, and determine the first cryoEM structure of a family member. Since these are very small membrane proteins, they leverage the antibodies developed for an inserted MPER peptide as fiducials. They show that the FEX channels are also channels that adopt an inverted topology. They also find that the overall architecture is similar to that of the Flucs, with a conserved Phe-box in the selectivity filter and a Na binding site in the center of the inverted repeat. However, they also show that the fluoride permeation pathway of CA- FEX is different from that of the Flucs, and that the extracellular vestibule is strongly electronegative, which could serve to lower fluoride flux. Using functional assays and MD simulations they show that in FEX channels, unlike in the Flucs, the Na site can be exchanged with the extracellular solution, suggesting it plays a modulatory role rather than a structural one. Overall, this is an excellent manuscript, well written, the experiments are well designed, carefully executed and properly interpreted. I have no major concerns, and have only some minor requests that the authors should consider, and some suggestions to improve clarity of presentation. My three small requests: 1) The MD simulations supporting the idea that a Na+ binding site is present in the central cavity of FEX are interesting. However, some controls are needed to support the authors’ conclusions that “these simulations demonstrate that the cation niche provides a binding site for sodium ion that is accessible to the external vestibule.” Indeed, no Na+ unbinding events are observed on the time scale of the simulations; which could be expected if the site is accessible to the extracellular solution and has millimolar affinity. Could the authors carry out simulations where they replace Na+ with K+? Do the K+ ions leave on the time scale of the simulations? Similarly, the observation that Li+ does not support function, would be easily testable in MD simulations. 2) The authors (Pg. 12) speculate that homologue-specific properties of different FEX family members arise from specific differences in the TM2/3 linker length and on the presence of a specific Trp side chain. This is an interesting hypothesis that could be readily tested by making mutations to lengthen or shorten the linker, or swap them between different homologues. I think it would be great if the authors could try at least 1-2 constructs to see if they express and if they can manipulate the conductance of the channels in this way. 3) In the discussion (Pg. 13-14), the authors formulate a complex hypothesis on the physiological role of the Na binding site in the FEX channel and about its voltage dependence. I appreciate the hypothesis. However, there is no evidence supporting it, or even of the fact that Na binding to this site is actually voltage dependent. I find the hypothesis to be too detailed and precise give the lack of evidence. I would suggest paring this down or completely removing it. Minor suggestions to improve clarity The use of the MPER peptide-FAB for cryoEM structure determination is a clever application. Could the authors elaborate a bit more on how they chose the MPER peptide insertion site, and/or if they tried other sites too? This would be helpful to the community to better understand strategies to use this approach to other proteins. Pg. 6, Fig. 2C The authors state “FEX possesses internal C2 pseudosymmetry with the N- and C-terminal domains tilted along a symmetry axis approximately 10° off the membrane normal (Figure 2C).” I find this statement a bit confusing; the dashed line in Fig. 2C is close to the plane of the membrane, not to the membrane normal. Pg. 7, Fig. 2C The authors state “A non-protein density consistent with a lipid or a detergent molecule segregates the linker helix, TM5, from the protein core, so that the core and linker are limited to a single point of contact: one putative hydrogen bond between S235 in the linker helix and H80 in the N-terminal core domain (Figure 2C).” I don't see the extra density in Fig 2C, and the level of detail described here of a single hydrogen bond cannot be appreciate in an overview panel. Please add close-up views to illustrate this point. Pg 4, Fig 1B. Please show -maybe in a supplementary panel- a bar chart with the averages and individual data points for the unitary transport rates of the homologues shows. Pg. 5, Fig. 1C. The authors could add some dashed lines to the traces to indicate the levels of the jump and maybe move the scale bar to the side to help appreciate how small the channels are. Pg. 5, “The measured reversal potentials were Nernstian with respect to the fluoride activity gradient (Figure 1F). This experiment provides unequivocal evidence of an electrodiffusive ion transport mechanism.” This is not correct. A uniporter mechanism (i.e., facilitated diffusion) would also be consistent with a Nernstian behavior without electrodiffusion. Pg 2, Introduction. “…and at least three types of membrane protein…” should be “and at least three types of membrane proteins…”", "reviewer_2": "The paper by Kang et al. reports the structural and initial functional characterization of members of the FEX family of fluoride export proteins. The authors resolve a 3D structure of a FEX protein from pathogenic yeast Candida albicans using cryo-EM. Moreover, they use purification and reconstitution to describe the fluoride transport properties of FEX proteins from Candida albicans, Arabidopsis thaliana, and S. cerevisiae. Fluoride transport is of high physiological importance, and thus far the molecular function of fluoride channels and transporters remains insufficiently understood, so that the results of this paper are of high importance. The Stockbridge group is leader in studying such proteins, and the paper greatly profits from the multiple experimental tools they developed to study these intriguing proteins. I have several concerns and suggestions for improvements. 1. Functional characterization 1.1. The functional characterization of the FEX proteins is based on F- efflux experiments with vesicles with reconstituted FEX and electrophysiological characterization in planar lipid bilayers. The authors estimate the unitary current by changes in holding currents after vesicle fusion in lipid bilayer experiments and obtain from these experiments a unitary conductance of 3 pS (Fig. 1C). I would assume that the unitary current is the minimum step observed in such experiments, and not the peak in such histograms, since more than one channel could be incorporated in the fused vesicle. Can one be sure to see fusions with only one channel? What if only vesicles with several channel fuse? In this case, the unitarty current would be overestimated. Since this experiment is important in the functional analysis, please enlarge the representative recordings. 1.2. Although it appears in principle possible, I am not aware of any ion channels that is permanently open, without closure. Since FEX channel function depends on Na+, Na+ binding and unbinding should induce gating the channels. The author should test whether such a behavior (which is a central conclusion of the paper) can be observed in planar bilayer recordings. 1.3. I am not so familiar with fluoride channels; I would assume that fluoride channels can also conduct other anions. How selective are the FEX channels, can they conduct Cl- or I-? 1.4. The authors mention that the Flucs, but not the FEXs have double-barreled pores. Whereas the structural basis of the double-barreled behavior of the CLCs is very obvious, I donot remember it for the Flucs. Could the authors improve the illustration of this behavior in Flucs and how it is abolished in the FEXs? 2. Molecular Simulations 2.1. Since the FEXs are a fluoride channels, the simulation should not be performed with 150 mM NaCl, but with Fluoride. I know about the problems in fluoride parameterization for atomistic simulatoions, but these problems arise from differences in the fluoride chemistry (the much higher pK a of HF than of HCl) that play a role in FEX channels, especially when the authors suggest that Na+ can bind and unbind from FEXs in the surface membrane. 2.2. The experiments predict that K can partially substitute for Na, with 50% FEX activity upon complete substitution of Na by K. Please provide the KDs for Na and K and the binding selectivity of the binding of the Na binding site. 2.3. Does the FEX channel undergo conformational changes upon Na/K binding, visible as changes in RSMDs? 3. Conclusion I read the final part of the paper as if the authors propose that FEXs conduct exclusive fluoride outward currents because of the voltage dependence in Na+ binding. I agree that this is an attractive idea, however, it is basically not supported by experimental or simulation data. The authors could show in bilayer experiments that current directs/rectification depends on the presence of Na+ on one site (channel insertion in both directions makes such an experiment more complicated, but not impossible). I describe another possible experiment in 1.2. ´How fast activate the channels upon exchanging solutions from K+ to Na+-based?", "reviewer_3": "The manuscript titled “The molecular basis of sodium-dependent fluoride export by the eukaryotic fluoride channel FEX” by Chia-Yu Kang et al. presents a timely report on the mechanism of F– transport through eukaryotic F– channels (FEXs). This study reveals several key structural features of the FEX from Candida albicans (FEX-CA), including selective inactivation of one F– permeation pores, rerouting of the active pore, increased electronegativity of the external vestibule, and the presence of an exchangeable Na+ ion. These insights are supported by a combination of cryo-EM, electrophysiology, cell-based assays, and molecular dynamics (MD) simulations, making this work more informative than a purely structural report. Molecular dynamics (MD) simulations are well performed and documented. The manuscript is well organized and clearly written. I strongly support its publication in Nat. Commun., pending minor clarifications as outlined below. 1. I am curious about the diversity of F– transport rates across FEX homologs. The authors attributes variability in transport rates to differences in the TM2/3 linker but offers limited discussion. 1) While only FEX-CA has been experimentally resolved, could homologous models (e.g., via AlphaFold or homology modeling) be built for other FEXs like FEX-CS? Comparative analysis may support the proposed role of the TM2/3 linker and shed light on pore architecture. 2) The hypothesis seems to assume that F– accumulation at the channel surface is the rate-limiting step and that permeation energetics/kinetics are conserved across FEXs. However, the rates of both steps remain unquantified. Standard MD can help estimate binding kinetics, while free energy calculations can probe permeation barriers and rates. Both would need polarizable force fields. While I’m not suggesting new simulations, these methods may guide future investigations. 2. About Na+ exchange and vestibule electronegativity. 1) The term “exchangeable” suggests dynamic ion exchange through the external vestibule (Fig. 5). Is there direct evidence that this pathway supports Na+ exchange? The FEX-CA structure shows a solvent-accessible Na+-binding motif, in contrast to the buried motif in Fluc. However, static dehydration doesn’t preclude dynamic hydration during MD, as shown in Fluc simulations (Yue et al., Biophys. J. 2022, 121, 1336–1447). Could the apparent exchangeability arise from different Na+ affinities (i.e., Kd values) compared to Fluc (McIlwain et al., J. Mol. Biol. 2020, 432, 1098–1108)? 2) Do other FEX homologs, such as FEX-CS, also have an electronegative external vestibule?"}, "author_response": "Point-by-point response to comments raised by the reviewers\nWe thank the reviewers for their thoughtful comments. All three reviewers inquired about the\nkinetics of cation exchange. We have added new data addressing this. Because of several\ntechnical challenges, we could not measure exchange using electrophysiological experiments as\nsuggested, but we were able to monitor cation exchange in proteoliposome-based experiments\n(Figure 4D). We also performed new simulations with K+, which does leave the binding site on\nthe timescale of the simulation, and provides a nice counterpoint to our Na+ results (Figure 4F,\n4G, 4J).\nWe have also added a new experiment to test transport of several different halides and\npseudohalides (Supplementary Figure 4), added some AlphaFold analysis of other homologues\nas requested (Supplementary Figure 19 and 20), and performed simulations in a system\nsolvated with NaF rather than NaCl (Supplementary Figure 18).\nA point-by-point response to all comments follows.\nReviewer #1:\n1. The MD simulations supporting the idea that a Na+ binding site is present in the central\ncavity of FEX are interesting. However, some controls are needed to support the authors’\nconclusions that “these simulations demonstrate that the cation niche provides a binding site for\nsodium ion that is accessible to the external vestibule.” Indeed, no Na+ unbinding events are\nobserved on the time scale of the simulations; which could be expected if the site is accessible to\nthe extracellular solution and has millimolar affinity. Could the authors carry out simulations\nwhere they replace Na+ with K+? Do the K+ ions leave on the time scale of the simulations?\nSimilarly, the observation that Li+ does not support function, would be easily testable in MD\nsimulations.\nResponse:\nAlthough the binding constant is ~0.5 mM we think the ion is kinetically trapped and dissociates\nslower than a typical mM binding process. To address this experimentally, we have now\nexamined the rate of cation exchange. For technical reasons, we monitored Na+/Li+ exchange\nrather than Na+/K+ exchange (background K+ is required for our assays) or Na+ dissociation (due\nto the same background K+ issue). Replacement of Li+ with Na+ is slow (t of ~2 hours), but this\n1/2\nis an appropriate physiological timescale for yeasts and plants. We are including this data in the\nmanuscript as Figure 4D, and have added the following paragraph:\nTo approximate the kinetics of cation exchange, we performed a Li+/Na+ exchange experiment,\nin which we reconstituted FEX-CA proteoliposomes with 0.6 mM Li+. We then added 10 mM Na+\nto the experimental buffer and monitored the fractional activity of the sample as a function of\ntime. In these experiments, fluoride efflux increases over time, corresponding to substitution of\nthe initial Li+, which suppresses activity, with Na+, which supports activity (Figure 4D). This\nexperiment provides support for reversible cation binding to the protein in lipid membranes. The\nt for exchange of 2 hours suggests that the rate constants for cation association and\n1/2\ndissociation occur on a tens of minutes-to-hour timescale.\nWe also performed MD simulations in which we replaced the Na+ ion with K+. Within the\ntimescale of our simulations, we observed that K+ binding is weaker than that of Na+ and tends to\nmove out of the binding motifs. We are including this data in the manuscript Figure 4, and have\nadded the following paragraph:\nIn contrast, when simulations were performed with the weakly interacting K+, the cation was\nheld more loosely and tended to move out from the binding motif. The average RMSD of FEX is\nsimilar in presence of both cations, but the RMSD for K+ is larger than that of Na+, reflecting\ngreater displacement of K+ (Figure 4F). Figure 4G tracks the cations’ movement along the Z-\nand X-axes, and illustrates movement of K+ toward the external vestibule.\nAnd the following sentence:\nCompared to Na⁺, K⁺ is surrounded by more water (Figure 4K), which could contribute to its\ndisplacement from the niche.\n2. The authors (Pg. 12) speculate that homologue-specific properties of different FEX\nfamily members arise from specific differences in the TM2/3 linker length and on the presence\nof a specific Trp side chain. This is an interesting hypothesis that could be readily tested by\nmaking mutations to lengthen or shorten the linker, or swap them between different homologues.\nI think it would be great if the authors could try at least 1-2 constructs to see if they express and\nif they can manipulate the conductance of the channels in this way.\nResponse: Thank you for reviewer’s suggestion. We agree that it would be valuable to swap the\nTM2/3 loop from fast eukaryotic FEX to a slower one to see if that enhances the transport rate.\nHowever, it has been surprisingly (frustratingly…) difficult to introduce mutations without\ndisrupting protein expression. Even point mutations to the loop (including mutation to the\ntryptophan, W127A) disrupt expression to such an extent that we don’t observe fluoride\nresistance activity in yeast, much less purify any protein.\nReviewer 3 had a similar comment, and suggested an AlphaFold analysis of the 2/3 linker. We\nhave included this as Supplementary Figure 20. We also softened the text to make it clear that\nthe idea is speculative based on a structural correlation. We hope this is sufficient to address this\nreviewer’s comment too.\n3. In the discussion (Pg. 13-14), the authors formulate a complex hypothesis on the\nphysiological role of the Na binding site in the FEX channel and about its voltage dependence.\nI appreciate the hypothesis. However, there is no evidence supporting it, or even of the fact that\nNa binding to this site is actually voltage dependent. I find the hypothesis to be too detailed and\nprecise give the lack of evidence. I would suggest paring this down or completely removing it.\nResponse: As suggested, we have pared this down. The section now reads:\nThe kinetics of cation exchange are slower than many familiar ion channel gating processes.\nHowever, we speculate that reversible Na+ binding is nonetheless relevant to FEX function under\nphysiological circumstances, since microbial responses to environmental or metabolic stress\noccur on the hour timescale or longer, including ion channel mediated processes and membrane\npotential fluctuations (52, 53).\n4. The use of the MPER peptide-FAB for cryoEM structure determination is a clever\napplication. Could the authors elaborate a bit more on how they chose the MPER peptide\ninsertion site, and/or if they tried other sites too? This would be helpful to the community to\nbetter understand strategies to use this approach to other proteins.\nResponse: We have added some text to the methods to describe how we selected this fusion site.\nWe also described bioinformatic and biochemical procedures for assessing the MPER fusion site\nin detail in a previous publication, PMID: 33676924. We started this project before AlphaFold\nwas a thing (yes, it’s been that long)…the best approach to predicting the epitope fusion site now\nwould probably involve using AlphaFold.\nTo predict the best site of fusion for the MPER epitope tag, we prepared a homology model of the\nN-terminal FEX domain based on the Fluc-Ec2 structure and appended the tag at the position\nthat corresponded to previously determined structures of MPER-Fluc-Ec2 in complex with\nantibody(39). We assessed two antibody fragments, 10E8v4 and VRC42, for binding to this\nMPER-FEX-CA construct, and selected 10E8v4 based on the monodispersity of the complex in\nsize exclusion chromatography.\n5. Pg. 6, Fig. 2C The authors state “FEX possesses internal C2 pseudosymmetry with the N-\nand C-terminal domains tilted along a symmetry axis approximately 10° off the membrane\nnormal (Figure 2C).” I find this statement a bit confusing; the dashed line in Fig. 2C is close to\nthe plane of the membrane, not to the membrane normal.\nResponse: Thanks for catching that mental lapse. We updated the text to refer to the plane of the\nmembrane. We also updated Figure 2C to provide a clearer image of the calculated membrane\nboundaries.\n6. Pg. 7, Fig. 2C The authors state “A non-protein density consistent with a lipid or a\ndetergent molecule segregates the linker helix, TM5, from the protein core, so that the core and\nlinker are limited to a single point of contact: one putative hydrogen bond between S235 in the\nlinker helix and H80 in the N-terminal core domain (Figure 2C).” I don't see the extra density in\nFig 2C, and the level of detail described here of a single hydrogen bond cannot be appreciate in\nan overview panel. Please add close-up views to illustrate this point.\nResponse: Thank you for the suggestion. We have added a new supplementary figure\n(Supplementary Figure 11) to show segmented cryo-EM maps of FEX-CA and the non-protein\ndensity, as well as a cartoon representation of the point of contact between the linker helix and\nthe protein core.\n7. Pg 4, Fig 1B. Please show -maybe in a supplementary panel- a bar chart with the\naverages and individual data points for the unitary transport rates of the homologues shows.\nResponse: We have included this as Supplementary Figure 3. For FEX-CS, which exceeds the\nresponse limit for the fluoride electrode, we have represented individual data points at the limit\nof detection. We also note that all replicates are included in the source data file.\n8. Pg. 5, Fig. 1C. The authors could add some dashed lines to the traces to indicate the\nlevels of the jump and maybe move the scale bar to the side to help appreciate how small the\nchannels are.\nResponse: Thank you for the suggestion. We have made edits to Figure 1C.\n9. Pg. 5, “The measured reversal potentials were Nernstian with respect to the fluoride\nactivity gradient (Figure 1F). This experiment provides unequivocal evidence of an\nelectrodiffusive ion transport mechanism.” This is not correct. A uniporter mechanism (i.e.,\nfacilitated diffusion) would also be consistent with a Nernstian behavior without electrodiffusion.\nResponse: Yes, you’re right. We’ve updated the sentence, which now reads, “This experiment\nprovides unequivocal evidence that fluoride permeation is not coupled with any other ion.”\nThe single channel fusions seem to rule out a uniporter, however, since they reflect a rate of\ntransport more rapid than could be achieved if a conformational change was required. We have\nalso added the sentence:\nThis rate of transport is faster than would be expected for a mechanism that requires a\nconformational change.\nReviewer #2:\nFunctional characterization\n1. The functional characterization of the FEX proteins is based on F- efflux experiments\nwith vesicles with reconstituted FEX and electrophysiological characterization in planar lipid\nbilayers. The authors estimate the unitary current by changes in holding currents after vesicle\nfusion in lipid bilayer experiments and obtain from these experiments a unitary conductance of 3\npS (Fig. 1C). I would assume that the unitary current is the minimum step observed in such\nexperiments, and not the peak in such histograms, since more than one channel could be\nincorporated in the fused vesicle. Can one be sure to see fusions with only one channel? What if\nonly vesicles with several channel fuse? In this case, the unitarty current would be overestimated.\nSince this experiment is important in the functional analysis, please enlarge the representative\nrecordings.\nResponse: This is a good point, and we acknowledge that without any closures or channel\nblockers, it is difficult to rule out multi-channel fusions. However, we still favor the explanation\nthat most of these fusion events represent single channels for two reasons. First, we prepared the\nexperiment to favor single channel fusions. From our bulk reconstitutions at similar protein\ndensities (i.e. liposome flux assays), we can estimate the average number of proteins per vesicle\nbased on the fraction of unoccupied vesicles (see PMID: 34059289 for details about the\nstatistical distribution). Second, the most convincing electrophysiological evidence is the\nperiodicity of our histogram. The main peak is at 2.5 pS, but you’ll note a second, smaller peak\nat double the conductance, 5 pS. The most reasonable explanation for this is that the largest\npopulation of vesicles that fuse possess one channel, and there is a small population with two\nchannels, and potentially a very small 3-channel population. The width of each peak in the\nhistogram represents normal variation about the mean -- either channel-to-channel variation, or\nexperimental error in making the conductance measurement since these are very small currents\nand near the limit of what we can see with normal bilayer noise.\nWe have updated the text to clarify this point. The paragraph now reads:\nThe rapid fluoride transport by FEX-CS enabled planar bilayer electrophysiology with the\npurified proteins. For liposomes reconstituted at a low protein:lipid ratio, such that each\nliposome is expected to bear 1-2 FEX proteins based on a statistical analysis of liposome\noccupancy(35), we observe fusion to the bilayer in extremely small, but discrete steps of ~0.5 pA\nat -200 mV under salt gradient conditions (Figure 1C). Like Fluc-Bpe, the single channel\ncurrents do not exhibit sub-states or closures, and are fully open at potentials from -200 to +200\nmV. A histogram of conductances measured for single insertion events obtained from different\nbilayers exhibits a major population with a mean of 2.2 pS, and a second, smaller population\ncentered at a value approximately twice as large (4.5 pS). These peaks most likely represent the\nfusion of singly- and doubly- occupied FEX-CS liposomes, yielding a mean single channel\nconductance of 2.2 pS.\n2. Although it appears in principle possible, I am not aware of any ion channels that is\npermanently open, without closure. Since FEX channel function depends on Na+, Na+ binding\nand unbinding should induce gating the channels. The author should test whether such a behavior\n(which is a central conclusion of the paper) can be observed in planar bilayer recordings.\nResponse: Although rare, the bacterial Fluc channels provide precedent for a constitutively open\nchannel – operating essentially an extremely selective leak pathway for fluoride. For FEX, we\nhave not been able to measure Na+ gating in planar lipid bilayers, but we think this is due to the\nkinetics of Na+ binding/unbinding. It would not be technically feasible to measure this in bilayer\nelectrophysiology experiments, due to several problems, including random orientation of the\nreconstituted protein and the very slow kinetics of this process. We instead performed an\nexperiment where we monitored exchange in liposomes– see our response to reviewer 1, point 1.\nThis is clearly not a fast-gating process in the way that we are familiar with for many ion\nchannels, but it is also appropriate for the proposed physiology in yeasts and plants, in which\nmembrane depolarizations last hours-to-days.\n3. I am not so familiar with fluoride channels; I would assume that fluoride channels can\nalso conduct other anions. How selective are the FEX channels, can they conduct Cl- or I-?\nResponse: There is one report in the literature that compares FEX-mediated fluoride and\nchloride efflux from liposomes using ion-specific electrodes. This paper does not quantify\nselectivity, but data in the paper suggest it is at least 1000-fold (Tausta, Berbasova et al. 2021;\nPMID: 33744970). Based on the reviewer’s suggestion, we thought that a more complete ionic\nselectivity series would be a useful addition to the literature, so we examined additional halides\nand pseudohalides for transport using solid-supported membrane (SSM) electrophysiology (Cl-,\nBr-, I-, OCN-, SCN-, and NO -). We detected no appreciable transport of these other anions by\nsensitive than the electrode assays cited above). We have added these results as Supplementary\nFigure 4. Iodide gave us a high background signal in all of our samples, including the protein-\nfree sample, so we omitted it from the analysis. We have updated the text with these ion\nselectivity series, which the paragraph reads:\nWe also screened additional halides and pseudohalides, including chloride, bromide, nitrate,\ncyanate, and thiocyanate, for transport by FEX-CA and FEX-CS using solid-supported\nmembrane (SSM) electrophysiology, which detects transient currents elicited by electrogenic\nbinding or ion transport into vesicles capacitively coupled to an electrode. In these experiments,\nonly fluoride perfusion evoked a robust capacitive current. These experiments set a lower limit of\n100-fold selectivity for fluoride over chloride, bromide, and nitrate for both FEX homologues.\nThe fluoride-induced currents are at least 15-fold larger than those elicited by cyanate and\nthiocyanate. Because cyanate and thiocyanate generate a non-specific signal even in protein-free\nsamples, this likely represents an underestimate of FEX’s true selectivity against these\npseudohalides (Supplementary Figure 4).\n4. The authors mention that the Flucs, but not the FEXs have double-barreled pores.\nWhereas the structural basis of the double-barreled behavior of the CLCs is very obvious, I do\nnot remember it for the Flucs. Could the authors improve the illustration of this behavior in Flucs\nand how it is abolished in the FEXs?\nResponse: The original structures of Flucs implied that two pores were present\n(PMID: 26344196), an idea that was later experimentally validated by Chris Miller’s group\n(PMID: 27449280).\nTo help illustrate this point, we have included an additional supplementary figure with a side-by-\nside rendering of the phenylalanine box in Fluc and equivalent residues in FEX as\nSupplementary Figure 12.\n5. The experiments predict that K can partially substitute for Na, with 50% FEX activity\nupon complete substitution of Na by K. Please provide the KDs for Na and K and the binding\nselectivity of the binding of the Na binding site.\nResponse: It is difficult to directly measure Na+ or K+ binding, but we can estimate K values\nd\nbased on the efflux experiments described in the manuscript. Liposome efflux experiments\nrequire using buffer with 300 mM K+. FEX-CA exhibits 50% activity in this condition, without\nadded Na+, so we estimate a K+ K of ~300 mM. On this background, we measure an apparent\nd\nNa+ K of 0.4 mM. If we assume a simple competition for a single site, then the actual K for Na+\nd d\nis ~0.2 mM. Therefore, this site is ~1500-fold more selective for Na+ over K+. Similar arithmetic\nand assumptions for FEX-CS indicate that this homologue is ~2000-fold selective for Na+ (~0.3\nmM) over K+ (~650 mM).\nWe have updated the text with these estimates, indicating that they are approximate:\nBased on the residual fractional activity in the absence of Na+, we estimate that FEX-CA and\nFEX-CS bind K+ with ≥500 mM affinity. Assuming that these cations compete for a single site,\nwe estimate that the K for Na+ in the absence of K+ is ~0.2 mM for FEX-CA and ~0.3 mM for\nd\nFEX-CS, and thus that the site favors Na+ over K+ by over three orders of magnitude.\nEvaluating these binding constants computationally would require free energy calculations that\nare beyond the scope of this work.\n6. I read the final part of the paper as if the authors propose that FEXs conduct exclusive\nfluoride outward currents because of the voltage dependence in Na+ binding. I agree that this is\nan attractive idea, however, it is basically not supported by experimental or simulation data. The\nauthors could show in bilayer experiments that current directs/rectification depends on the\npresence of Na+ on one site (channel insertion in both directions makes such an experiment more\ncomplicated, but not impossible). I describe another possible experiment in 1.2. ´How fast\nactivate the channels upon exchanging solutions from K+ to Na+-based?\nResponse: Thanks for this suggestion regarding the voltage dependence of Na+ binding. Given\nthe small size of the single channels, the aforementioned problem of random orientation, and the\nrelatively slow kinetics of the process, we were not able to perform these experiments. We\ntherefore altered the text of the discussion to remove the hypothesis about the voltage\ndependence of Na+ binding. The section now reads:\nThe kinetics of cation exchange are slower than many familiar ion channel gating processes.\nHowever, we speculate that reversible Na+ binding is nonetheless relevant to FEX function under\nphysiological circumstances, since microbial responses to environmental or metabolic stress\noccur on the hour timescale or longer, including ion channel mediated processes and membrane\npotential fluctuations (52, 53).\nMolecular simulations\n7. Since the FEXs are a fluoride channels, the simulation should not be performed with 150\nmM NaCl, but with Fluoride. I know about the problems in fluoride parameterization for\natomistic simulations, but these problems arise from differences in the fluoride chemistry (the\nmuch higher pK a of HF than of HCl) that play a role in FEX channels, especially when the\nauthors suggest that Na+ can bind and unbind from FEXs in the surface membrane.\nResponse: Thank you for the suggestion. To mimic physiological conditions, we usually perform\nsimulation with 150 mM of NaCl. Considering F concentration is at most in the low mM range, we\nused the usual protocol. However, according to the reviewer’s suggestion, we repeated our\nsimulation with 150 mM NaF and the results are similar within the current simulation timescale. We\nhave added this comparison in the manuscript to Supplementary Figure 18, and added the following\nsentence:\nIn addition, we prepared another system solvated in NaF, showing similar behavior for both\nsystems containing Na+ ion (Supplementary Figure 18).\n8. Does the FEX channel undergo conformational changes upon Na/K binding, visible as\nchanges in RSMDs?\nResponse: We performed simulations with Na+ and K+ in the binding site and tracked the\nmovement of cations (Figure 4G). These results combined with RMSD (Figure 4F) show that\nK+ ion is less stable than Na+ in the binding motifs and indeed leaves the binding site. However,\nour results show similar mean RMSD for FEX with Na⁺ versus K⁺. We have added the\ncomparison of these two ions to Figure 4 in the manuscript.\nReviewer #3\n1. I am curious about the diversity of F– transport rates across FEX homologs. The authors\nattributes variability in transport rates to differences in the TM2/3 linker but offers limited\ndiscussion. While only FEX-CA has been experimentally resolved, could homologous models\n(e.g., via AlphaFold or homology modeling) be built for other FEXs like FEX-CS? Comparative\nanalysis may support the proposed role of the TM2/3 linker and shed light on pore architecture.\nResponse: Thank you for the suggestion. We used AlphaFold3 to predict 12 models of FEX\nhomologues covering a range of eukaryotic clades and used these to compare the predicted\nstructure of the TM2 and TM3a helices (Supplementary Figure 20). The length and orientation\nof TM2/3 loop vary among species compared to our FEX-CA model, which has a longer TM2/3\nloop, located near the entrance of the channel. In particular, FEX-CS, FEX-AT, and FEX-SC\nexhibited faster unitary turnover rates and possess a shorter, helical TM2/3 linker, suggesting a\npotential relationship between the length and secondary structure of the TM2/3 linker and\ntransport kinetics. To better understand the variation in fluoride transport rates among species,\nfurther functional characterization would be required, such as comparing homologs with longer\nTM2/TM3 loop (i.e., FEX-CC) and with shorter TM2/TM3 loop (i.e., FEX-OB), as well as\nmutagenesis of FEX homologs.\nWe have updated the paragraph to include this result, and make it completely clear that this is\nspeculative based on correlation between the fast rate and shorter loops.\nIn the plant proteins, FEX-CS and FEX-AT, which exhibit currents sufficiently large for detection\nby single molecule electrophysiology, this linker is composed of a short -helix that lacks the\ntryptophan that extends most deeply into the vestibular volume. Based on this correlation, we\nspeculate that homologue-specific properties of the TM2/3 linker might be one factor that\ncontributes to the rate of fluoride export exhibited by different FEX homologues.\n2. The hypothesis seems to assume that F– accumulation at the channel surface is the rate-\nlimiting step and that permeation energetics/kinetics are conserved across FEXs. However, the\nrates of both steps remain unquantified. Standard MD can help estimate binding kinetics, while\nfree energy calculations can probe permeation barriers and rates. Both would need polarizable\nforce fields. While I’m not suggesting new simulations, these methods may guide future\ninvestigations.\nResponse: Thank you for reviewer’s insightful comments. Although these experiments are\nbeyond the scope of our current study, we think these potential approaches can guide future\ninvestigations and refine our comprehensive understanding of FEXs. Thank you again for\nhighlighting these promising avenues.\n3. The term “exchangeable” suggests dynamic ion exchange through the external vestibule\n(Fig. 5). Is there direct evidence that this pathway supports Na+ exchange? The FEX-CA\nstructure shows a solvent-accessible Na+-binding motif, in contrast to the buried motif in Fluc.\nHowever, static dehydration doesn’t preclude dynamic hydration during MD, as shown in Fluc\nsimulations (Yue et al., Biophys. J. 2022, 121, 1336–1447). Could the apparent exchangeability\narise from different Na+ affinities (i.e., Kd values) compared to Fluc (McIlwain et al., J. Mol.\nBiol. 2020, 432, 1098–1108)?\nResponse: We have obtained direct experimental evidence of cation exchange (see our response\nto reviewer 1, point 1, and Figure 4D). However, we do not have functional evidence this occurs\nthrough the external vestibule at this point. We think it is very likely, though, given the\narchitecture of the protein. We have added a comment to the results to describe that this\ninterpretation arises from the current structure:\nIn our structures, the niche motif appears to be at least partly exposed to the aqueous solution\nfrom the external vestibule, and inaccessible from the cytoplasmic side.\nSince we have only been able to detect reversible Na+ exchange in specific mutants of Fluc\n(McIlwain et al., JMB, PMID: 31945374), we do not have a good estimate for the Na+ K in WT\nd\nFluc. Simulations have suggested that in Fluc, dimer formation is a two-step process, where the\nmonomers initially interact in a weak complex (which allows Na+ to enter the binding site),\nfollowed by formation of a tight dimer that locks in Na+ at the interface (PMID: 37956279).\nThus, the fact that we can experimentally monitor any cation exchange in the WT protein is an\nimportant distinction between Fluc and FEX.\nIn addition, simulations show that the K⁺ ion leaves the binding motifs during the simulation,\nindicating it is weakly bound and can be replaced by Na⁺. We appreciate the recommendation to\ncompute Kd; however, that would necessitate free-energy calculations beyond our current\nexperimental scope.\n4. Do other FEX homologs, such as FEX-CS, also have an electronegative external\nvestibule?\nResponse: There is not a strong sequence conservation among the vestibule-lining residues, but\nwe have added some additional analysis of AlphaFold-predicted models drawn from a variety of\neukaryotic clades. These show that the external vestibule electrostatics are similarly negative for\nmany FEX homologues (new Supplementary Figure 19), including FEX-CS. There are\nexceptions, however. We have added the following sentence to the discussion:\nSequence analysis and structure prediction for other FEX homologues suggests that while\nspecific acidic amino acids are not conserved, the overall negative character of this vestibule is\npresent in diverse FEX homologues, with a few exceptions (Supplementary Figure 19)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all my queries and I recommend their manuscript for publication in Nature Communications.", "reviewer_2": "The authors have adressed most of the comments. I only remain unsatisfied by their comments on a question I asked about the Molecular simulations. “2.1. Since the FEXs are a fluoride channels, the simulation should not be performed with 150 mM NaCl, but with Fluoride. I know about the problems in fluoride parameterization for atomistic simulations, but these problems arise from differences in the fluoride chemistry (the much higher pKa of HF than of HCl) that play a role in FEX channels, especially when the authors suggest that Na+ can bind and unbind from FEXs in the surface membrane.“ The authors simply state “However, according to the reviewer’s suggestion, we repeated our simulation with 150 mM NaF and the results are similar within the current simulation timescale.“ I cannot find any details about these simulations. Which force field was used in these simulations, how did the authors deal with the unfavorable HF dissociation in water? I do not want to force the authors to perform QM/MM simulations, I would be fully satisfied by a comment that adressed the problems in studying F- permeation or binding in atomistic simulations.", "reviewer_3": "In the revised manuscript, the authors have addressed all my concerns. I'd like to endorse its publication in Nat. Commun."}, "author_response": "Response to comments raised by the reviewers\nWe thank the reviewers for their thorough review and recommendation to accept our\nmanuscript. We greatly appreciate your constructive feedback, which has significantly\nenhanced the quality of our work. Below, we provide a response to the Reviewer #2\ncomments and describe the corresponding changes made in the revised manuscript.\nReviewer #2 (Remarks to the Author):\nThe authors have adressed most of the comments. I only remain unsatisfied by their\ncomments on a question I asked about the Molecular simulations.\n“2.1. Since the FEXs are a fluoride channels, the simulation should not be performed with\n150 mM NaCl, but with Fluoride. I know about the problems in fluoride parameterization\nfor atomistic simulations, but these problems arise from differences in the fluoride\nchemistry (the much higher pKa of HF than of HCl) that play a role in FEX channels,\nespecially when the authors suggest that Na+ can bind and unbind from FEXs in the\nsurface membrane.\nThe authors simply state “However, according to the reviewer’s suggestion, we repeated\nour simulation with 150 mM NaF and the results are similar within the current simulation\ntimescale.“ I cannot find any details about these simulations. Which force field was used\nin these simulations, how did the authors deal with the unfavorable HF dissociation in\nwater?\nI do not want to force the authors to perform QM/MM simulations, I would be fully satisfied\nby a comment that adressed the problems in studying F- permeation or binding in\natomistic simulations.\nWe thank the reviewer for raising this important point regarding the challenges of\nmodeling fluoride in classical MD simulations. We agree that treating fluoride accurately\nis challenging since it has a very high charge density and strong hydration, and its\nbehavior is known to be difficult to capture with non-polarizable force fields. We have\nadded a concise statement in the revised manuscript acknowledging the known\nlimitations of modeling fluoride with non-polarizable force fields and clarifying the rationale\nfor the simulation setup.\n“It is important to note that accurate atomistic modeling of F⁻ permeation or binding is\nintrinsically challenging due to its high charge density and strong hydration, which might\nnot be fully captured by standard non-polarizable force fields. Accordingly, our simulations\nwere not designed to resolve detailed fluoride transport energetics or permeation\npathways.”\nRegarding the reviewer’s question about HF dissociation in water and the challenges of\nmodeling HF with non-polarizable force fields: In our simulations, HF is not explicitly\nincluded. At physiological pH, HF is predominantly present as H+ and F⁻, so there is no\nor very small number of HF in the system based on Henderson-Hasselbalch equation.\npH= pKa + log [F-]/[HF] = 10 pH - pKa = [F-]/[HF] = 10 (7.4 – 3.17) = [F-]/[HF]\nFor every 1 molecule of HF, there are ~17,000 F⁻ ions at pH ~7.4. Hence, fluoride is\npresent almost entirely as F⁻ rather than HF. Because hydronium ion cannot be reliably\nmodeled with a non-polarizable force field, the system was modeled with NaF rather than\nHF.\nFull details of all simulations and the applied force fields are identical across systems and\nare provided in the Methods section:\n“The starting structure of FEX-CA was generated using the newly determined cryo-EM\nstructure (PDB: 9OP1). This structure was aligned with Fluc-Bpe (PDB: 5NKQ) to place\nthe Na+ or K+ ion in the niche. Afterwards, the protonation states of all titratable residues\nat pH 7.4 were determined using the H++ web server69,70. The protein was inserted into\na 3:1 POPE/POPG bilayer and solvated with TIP3P71 water molecules using Packmol-\nMemgen72. Missing hydrogen atoms and Na+ ions were added to neutralize the system,\nand NaCl, NaF, or KCl ions were then added using the tleap module from the\nAmberTools2073 to achieve a physiological salt concentration of 0.150 M. All systems\nconsisted of 98,092 atoms. The initial coordinate and parameter files were generated\nusing tleap73, applying the Amber ff14SB74, lipid 2175, and TIP3P71 forcefields.”\nWe added a sentence here to make it more clear about the number of simulations:\n“Three independent simulation trials were performed using the pmemd.cuda module of\nAmber20 software package72,76 for each of the systems solvated in NaCl, NaF or KCl\nto ensure adequate sampling.”\nWe hope that these additions address the reviewer’s concerns and clarify both the\nlimitations and the rationale behind our simulation setup."}]

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