Abstract
Fluc family fluoride channels protect microbes against ambient environmental fluoride by undermining the cytoplasmic accumulation of this toxic halide. These proteins are structurally idiosyncratic, and thus the permeation pathway and mechanism have no analogy in other known ion channels. Although fluoride-binding sites were identified in previous structural studies, it was not evident how these ions access aqueous solution, and the molecular determinants of anion recognition and selectivity have not been elucidated. Using x-ray crystallography, planar bilayer electrophysiology, and liposome-based assays, we identified additional binding sites along the permeation pathway. We used this information to develop an oriented system for planar lipid bilayer electrophysiology and observed anion block at one of these sites, revealing insights into the mechanism of anion recognition. We propose a permeation mechanism involving alternating occupancy of anion-binding sites that are fully assembled only as the substrate approaches.
Highlights
Microbes are protected from the cytoplasmic accumulation of environmental fluoride ion by export of the toxic anion via fluoride channels known as Flucs [1,2]
We fuse electrophysiology, X-ray crystallography, and liposome flux assays to identify the routes by which fluoride ions access the previously identified fluoride binding sites along the polar track of Fluc homologues Fluc-Bpe and Fluc-Ec2
Conversion of a serine from this anion binding site to a cysteine introduces a strong pH-dependence to the fluoride channel activity, demonstrating that this position comprises part of the permeation pathway
Summary
Microbes are protected from the cytoplasmic accumulation of environmental fluoride ion by export of the toxic anion via fluoride channels known as Flucs [1,2]. These small, homodimeric ion channels are remarkable proteins in two regards: first, their unusual “dual topology” architecture, in which the two subunits of the homodimer are arranged antiparallel with respect to each other [3,4], yielding a double-barreled pair of pores related by two-fold symmetry [5,6,7,8]. In contrast to most characterized families of anion channels, which tend to be non-selective among anions, and sometimes poorly discriminate against cations, the Flucs are arguably the most selective ion channels known, with >10,000 fold selectivity against the biologically abundant chloride [4]. Most characterized anion channels handle the most abundant ion in their milieu, ususally chloride ion, and other halides and pseudohalides that might compete with the physiological ion are present at much lower concentrations
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