The fluoride permeation pathway and anion recognition in Fluc family fluoride channels.

For all of Fluc’s idiosyncrasies, we propose a mechanism with much in common with other well-characterized ion channels (Figure 5). The negative charge of the fluoride ions is counterbalanced by the protein’s few positive charges, the vestibule arginines and the structural central Na⁺. Experiments have shown that both pores are functional for F^– permeation (Last et al., 2016), but it seems highly unlikely that all six anion positions (three anions in each of two pores) are simultaneously occupied. Rather, we imagine a scenario of alternating occupancy, as proposed for other multi-ion pores, in which a fluoride moving into one binding site electrostatically hastens its neighbor into the next position in the sequence. We propose either that the densities observed in the crystal structure represent partially occupied fluoride sites or that the monobodies used as crystallization chaperones alter the electrostatic landscape in the pore, increasing ion occupancy. Indeed, in crystal structures of Fluc-Bpe with a monobody occupying only one side of the channel, each pore contained only one fluoride density, rather than two, in the polar track (McIlwain et al., 2018).

In Figure 5, the starting configuration (left panel) shows an F^– bound in the site identified by anomalous scattering, at the bottom of the vestibule, labeled F0. We propose that as additional fluoride ions enter the electropositive vestibule, the fluoride ion at F0 is electrostatically repelled, providing energy for desolvation and translocation into the narrowest part of the channel at position F1 (Figure 5, right panel). But the F1 binding site is not pre-assembled: rotamerization of the vestibule serine (S83 in Fluc-Bpe), which is possible with serine but not threonine, accompanies the lateral movement of the anion. Other sidechains have also been proposed to adopt new rotameric conformations in order to ligand the anion at F1, including N43 (Stockbridge et al., 2015) and S84 (Last et al., 2017). Thus, we propose that the F1 binding site is assembled simultaneously with its occupation by fluoride. The rotamerization of channel sidechains to accompany ions through the pore has been proposed for other channels as well, including the Orai and voltage-gated calcium channels (Hou et al., 2020; Sather and McCleskey, 2003).

We imagine that this configuration is short-lived: a new fluoride ion settles into the deep vestibule F0 site, the fluoride at F1 moves farther down the channel to F2, and the S83 sidechain returns to its position facing the vestibule. The binding site at F2 is in close proximity to the E88 carboxylate; the electrostatic conflict could be resolved if E88 swings out into solution, allowing the fluoride at F2 to exit the channel, having now traversed the bilayer (whereby E88 could then resume its position at the pore exit without conflict). We have shown that the E88Q mutant reduces both fluoride currents and block of fluoride currents by OCN^–. We propose that both behaviors arise because the mutant sidechain, which does not bear a negative charge, is not easily dislodged from the binding site via electrostatic conflicts with the permeant fluoride or the cyanate blocker.

This proposed mechanism introduces several previously unrecognized amino acids involved in fluoride permeation and extends the pathway to the aqueous solutions on both sides of the bilayer. It also explains the evolutionary conservation and physiological consequences of mutation described for conserved sidechains, including the invariant serine (position S81 in Ec2 or S83 in Bpe) and triad glutamate (position E88 in Bpe) (Smith et al., 2015; Berbasova et al., 2017). Also, while these experiments provide the first hints of a molecular mechanism for anion recognition by the Flucs, they also emphasize how robust the channel’s anion selectivity is. Despite dozens of point mutations to two homologs, alone and in combination (summarized in Figure 5—figure supplement 1 and Table 4), no mutant that permits the permeation of any other anion has been reported yet. It may be that there is no unique selectivity filter but that several regions of the channel work together to achieve selectivity, so that abolishing anion selectivity requires destruction of the channel itself. Alternatively, channel selectivity might be achieved by matching the number of available ligands in the pore to the preferred coordination number of the anion, as has been proposed for K⁺ channels (Bostick and Brooks, 2007; Bostick and Brooks, 2009). F^– is a superlative in this regard, requiring fewer ligands than any other anion. If this is the case, relaxing the selectivity might require adding coordinating ligands along the pore, which would be difficult to accomplish with site-directed mutagenesis alone. Indeed, even accounting for the addition of coordinating ligands via sidechain rotamerization, the F1 and F2 sites have relatively small coordination numbers (~four including the phenylalanine ring edges). Chloride, in contrast, prefers at least six ligands in its coordination sphere (Bostick and Brooks, 2009; Ohtaki and Radnai, 1993; Cametti and Rissanen, 2009; Merchant and Asthagiri, 2009).