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

Chloride homeostasis is regulated in all cellular compartments. CLC-type channels selectively transport Cl⁻ across biological membranes. It is proposed that side-chains of pore-lining residues determine Cl⁻ selectivity in CLC-type channels, but their spatial orientation and contributions to selectivity are not conserved. This suggests a possible role for mainchain amides in selectivity. We use nonsense suppression to insert α-hydroxy acids at pore-lining positions in two CLC-type channels, CLC-0 and bCLC-k, thus exchanging peptide-bond amides with ester-bond oxygens which are incapable of hydrogen-bonding. Backbone substitutions functionally degrade inter-anion discrimination in a site-specific manner. The presence of a pore-occupying glutamate side chain modulates these effects. Molecular dynamics simulations show backbone amides determine ion energetics within the bCLC-k pore and how insertion of an α-hydroxy acid alters selectivity. We propose that backbone-ion interactions are determinants of Cl⁻ specificity in CLC channels in a mechanism reminiscent of that described for K⁺ channels.