Backbone Amide Nitrogen Atoms are Key Determinants of Inter-Anion Discrimination in CLCs

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Most known Cl- channels are more permeable to other anions, such as I- or NO3-, than to their namesake Cl- ion. CLC channels, in contrast, are more permeable to Cl- and have a unique selectivity sequence of Cl->Br->NO3->I-, suggesting that the mechanism of ion selectivity in the CLCs is different from that of other Cl- channels. It has been proposed that CLC selectivity is primarily determined by interactions of pore-lining side chains with the permeating ions. Specifically, a serine residue (Sercen) which participates in the central Cl- binding site was shown to control the Cl- vs NO3- selectivity of CLC channels and transporters. Surprisingly, in the structure of the ClC-K channel, which follows the signature CLC selectivity sequence, Sercen points away from the pore. Here, we show that mutations at Sercen do not alter the selectivity sequence of the CLC-K channel, suggesting that this residue is not a general determinant of CLC selectivity. Since all CLCs share a common selectivity sequence, we hypothesize that the backbone amides, which coordinate the ions in the pore, might be the conserved determinants of selectivity. To test this possibility directly, we used atomic-scale mutagenesis with non-canonical amino acids to replace pore-lining amide nitrogen atoms with oxygen atoms, by introducing α-hydroxy acids in ClC-0 and ClC-K. This maneuver eliminates the hydrogen bonding capability of the backbone NH groups while leaving the side-chain composition unperturbed. We found that CIC-0 and CIC-K channels with individual backbone substitutions displayed macroscopic currents in Xenopus laevis oocytes with a strongly degraded inter-anionic selectivity. The data suggests that the pore-lining backbone amides are the general determinants of inter-anion selectivity in CLCs and that side chains contribute to selectivity in only a subset of CLCs.