Backbone amides are determinants of Cl− selectivity in CLC ion channels
Lilia Leisle, Kin Lam, Sepehr Dehghani‐Ghahnaviyeh, Eva Fortea, Jason D. Galpin, Christopher A. Ahern, Emad Tajkhorshid, Alessio Accardi
Abstract
Abstract 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.