F-/Cl- selectivity in CLCF-type F-/H+ antiporters.

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.

Surprises from an unusual CLC homolog.

The chloride channel (CLC) family is distinctive in that some members are Cl(-) ion channels and others are Cl(-)/H(+) antiporters. The molecular mechanism that couples H(+) and Cl(-) transport in the antiporters remains unknown. Our characterization of a novel bacterial homolog from Citrobacter koseri, CLC-ck2, has yielded surprising discoveries about the requirements for both Cl(-) and H(+) transport in CLC proteins. First, even though CLC-ck2 lacks conserved amino acids near the Cl(-)-binding sites that are part of the CLC selectivity signature sequence, this protein catalyzes Cl(-) transport, albeit slowly. Ion selectivity in CLC-ck2 is similar to that in CLC-ec1, except that SO(4)(2-) strongly competes with Cl(-) uptake through CLC-ck2 but has no effect on CLC-ec1. Second, and even more surprisingly, CLC-ck2 is a Cl(-)/H(+) antiporter, even though it contains an isoleucine at the Glu(in) position that was previously thought to be a critical part of the H(+) pathway. CLC-ck2 is the first known antiporter that contains a nonpolar residue at this position. Introduction of a glutamate at the Glu(in) site in CLC-ck2 does not increase H(+) flux. Like other CLC antiporters, mutation of the external glutamate gate (Glu(ex)) in CLC-ck2 prevents H(+) flux. Hence, Glu(ex), but not Glu(in), is critical for H(+) permeation in CLC proteins.