1.A.43 The Camphor Resistance or Fluoride Channel (Fluc) Family
CrcB of E. coli appears to confer camphor resistance when overexpressed and induces resA expression (Sand et al. 2003). Deletion is not lethal but increases sensitivity to camphor and fluoride. CrcB has 127 aas and 4 TMSs. Its many close homologues are small proteins, usually with 4 TMSs. These proteins mediate fluoride efflux and resistance (Ji et al. 2014). The active transporter is a dimer of 4 TMS subunits arranged antiparallel (Stockbridge et al. 2014).
Fluc family fluoride channels are assembled as primitive antiparallel homodimers (McIlwain et al. 2020). Crystallographic studies revealed a cation bound at the center of the protein, where it is coordinated at the dimer interface by mainchain carbonyl oxygens from the mid-membrane breaks in two corresponding TMSs. This cation is a stably bound sodium ion, and, although it is not a transported substrate, its presence is required for the channel to adopt an open, fluoride conducting conformation. The interfacial site is selective for sodium over other cations, except for Li+, which competes with Na+ for binding, but does not support channel activity. Fluoride, F-, ubiquitous in soil, water and marine environments, is a chronic threat to microorganisms. Many bacteria, archaea, unicellular eukaryotes and plants use F- exporters to lower cytoplasmic F- levels to counteract the anion's toxicity (McIlwain et al. 2020).
Stockbridge et al. 2013 and Li et al. 2013 showed that these ''Fluc'' proteins, purified and reconstituted in liposomes and planar phospholipid bilayers, form constitutively open anion channels with extreme selectivity for F- over Cl-. The active channel is a dimer of identical or homologous subunits arranged in antiparallel transmembrane orientation, or a single protein with two internal 4TMS repeats (as for the yeast proteins (TC# 1.A.43.2.4 and 5). This dual-topological assembly had not previously been seen in ion channels but is known in multidrug transporters of the SMR sub-family of the DMT superfamily (TC# 2.A.7.1) (Pornillos et al. 2005; Pornillos and Chang 2006).
Crystal structures showed that Fluc channels contain two separate ion-conduction pathways, each with two F- binding sites (Stockbridge et al. 2015). Last et al. 2016 examined the consequences of mutating two conserved F--coordinating phenylalanine residues. Substitution of each phenylalanine specifically extinguished its associated F- binding site in crystal structures and concomitantly inhibited F- permeation. Functional analysis of concatemeric channels, which permit mutagenic manipulation of individual pores, showed that each pore can be separately inactivated without blocking F- conduction through its symmetry-related twin. The results strongly supported a dual-pathway architecture of these channels (Last et al. 2016).