Many bacterial species protect themselves against environmental F(-) toxicity by exporting this anion from the cytoplasm via CLC(F) F(-)/H(+) antiporters, a subclass of CLC superfamily anion transporters. Strong F(-) over Cl(-) selectivity is biologically essential for these membrane proteins because Cl(-) is orders of magnitude more abundant in the biosphere than F(-). Sequence comparisons reveal differences between CLC(F)s and canonical Cl(-)-transporting CLCs within regions that, in the canonical CLCs, coordinate Cl(-) ion and govern anion transport. A phylogenetic split within the CLC(F) clade, manifested in sequence divergence in the vicinity of this ion-binding center, raises the possibility that these two CLC(F) subclades might exhibit differences in anion selectivity. Several CLC(F) homologues from each subclade were examined for F(-)/Cl(-) selectivity of anion transport and equilibrium binding. Differences in both of these anion-selectivity metrics correlate with sequence divergence among CLC(F)s. Chimeric constructs identify two residues in this region that largely account for the subclade differences in selectivity. In addition, these experiments serendipitously uncovered an unusually steep, Cl(-)-specific voltage dependence of transport that greatly enhances F(-) selectivity at low voltage.

Cl(-)/H(+) antiporters of the CLC superfamily transport anions across biological membranes in varied physiological contexts. These proteins are weakly selective among anions commonly studied, including Cl(-), Br(-), I(-), NO3(-) and SCN(-), but they seem to be very selective against F(-). The recent discovery of a new CLC clade of F(-)/H(+) antiporters, which are highly selective for F(-) over Cl(-), led us to investigate the mechanism of Cl(-)-over-F(-) selectivity by a CLC Cl(-)/H(+) antiporter, CLC-ec1. By subjecting purified CLC-ec1 to anion transport measurements, electrophysiological recording, equilibrium ligand-binding studies and X-ray crystallography, we show that F(-) binds in the Cl(-) transport pathway with affinity similar to Cl(-) but stalls the transport cycle. Examination of various mutant antiporters implies a 'lock-down' mechanism of F(-) inhibition, in which F(-), by virtue of its unique hydrogen-bonding chemistry, greatly retards a proton-linked conformational change essential for the transport cycle of CLC-ec1.

Anion channels and antiporters of the ClC superfamily have been found to be exclusively dimeric in nature, even though each individual monomer contains the complete transport pathway. Here, we describe the destabilization through mutagenesis of the dimer interface of a bacterial F(-)/H(+) antiporter, ClC(F)-eca. Several mutations that produce monomer/dimer equilibrium of the normally dimeric transporter were found, simply by shortening a hydrophobic side chain in some cases. One mutation, L376W, leads to a wholly monomeric variant that shows full activity. Furthermore, we discovered a naturally destabilized homologue, ClC(F)-rla, which undergoes partial monomerization in detergent without additional mutations. These results, in combination with the previous functional monomerization of the distant relative ClC-ec1, demonstrate that the monomer alone is the functional unit for several clades of the ClC superfamily.