Backbone amides are key determinants of Cl ⁻ selectivity in CLC ion channels

Chloride homeostasis is tightly regulated in cellular compartments by dedicated channels and transporters. Whereas CLC-type channels select for Cl ⁻ over other anions, all other ‘chloride’ channels are indiscriminate in their anionic permeability. Pore-lining side chains are thought to determine Cl ⁻ selectivity in CLC channels. However, orientation and functional roles of these side chains is not conserved among CLCs. All CLC pores are lined by backbone amides in a conserved structural arrangement, suggesting a role of mainchain groups in selectivity. We replaced pore-lining residues in the CLC-0 and bCLC-k channels with their respective α-hydroxy acid counterparts using nonsense suppression method. This exchanges peptide-bond amides with ester-bond oxygens, incapable of hydrogen-bonding with permeating anions. Backbone substitutions functionally degrade inter-anion discrimination in a site-specific manner. These effects depend on the presence of a glutamate side chain that competes with ions permeating through the pore. Molecular dynamics simulations show that ion energetics within the bCLC-k pore are primarily determined by interactions with backbone amides. Insertion of an α-hydroxy acid significantly alters ion selectivity and global pore hydration. We propose that backbone amides are conserved determinants of Cl ⁻ specificity in CLC channels in a mechanism reminiscent of that described for K ⁺ channels.